text:
Therapeutic Efficacy of a {sup 188}Re-Labeled {alpha}-Melanocyte-Stimulating Hormone Peptide Analog in Murine and Human Melanoma-Bearing Mouse Models
Miao, Yubin; Owen, Nellie K.; Fisher, Darrell R.; Hoffman, Timothy J.; Quinn, Thomas P.
The purpose of this study was to examine the therapeutic efficacy of {sup 188}Re-(Arg{sup 11})CCMSH in the B16/F1 murine melanoma and TXM13 human melanoma bearing mouse models. Method: (Arg11)CCMSH was synthesized and labeled with {sup 188}Re to form {sup 188}Re-(Agr{sup 11})CCMSH. B16/F1 melanoma tumor bearing mice were administrated with 200 Ci, 600 Ci and 2x400 Ci of {sup 188}Re-(Arg{sup 11})CCMSH via the tail vein, respectively. TXM13 melanoma tumor hearing mice were separately injected with 600 Ci, 2x400 Ci and 1000 Ci of 100Re-(Arg{sup 11})CCMSH through the tail vein. Two groups of 10 mice bearing either B16/F1 or TXM13 tumors were injected with saline as untreated controls. Results: In contrast to the untreated control group, {sup 188}Re(Arg11)CCMSH yielded rapid and lasting therapeutic effects in the treatment groups with either B16/F1 or TXM13 tumors. The tumor growth rate was reduced and the survival rate was prolonged in the treatment groups. Treatment with 2x400 Ci of {sup 188}Re-Arg{sup 11}CCMSH significantly extended the mean life of B16/F1 tumor mice (p<0.05), while the mean life of TXm13 tumor mice was significantly prolonged after treatment with 600 Ci and 1000 Ci doses of {sup 188}Re-(Arg{sup 11})CCMSH (p<0.05 High-dose {sup 188}Re-(Arg{sup 11}))CCMSH produced no observed normal-tissue toxicity. Conclusions: The therapy study results revealed that {sup 188}Re-Arg11 CCMSH yielded significant therapeutic effects in both B16/F1 murine melanoma and TXM13 human melanoma bearing mouse models. {sup 188}Re-(Arg{sup 11})CCMSH appears to be a promising radiolabeled peptide for targeted radionuclide therapy of melanoma.
Suppression of production of mouse 28S ribosomal RNA in mouse-human hybrids segregating mouse chromosomes.
Croce, C M; Talavera, A; Basilico, C; Miller, O J
Mouse-human somatic cell hybrids that lose (segregate) human chromosomes produce only mouse 28S ribosomal RNA even when they retain copies of the human chromosomes that contain the genes for 28S ribosomal RNA. In contrast, mouse-human hybrid cells that segregate mouse chromosomes produce only human 28S ribosomal RNA even when they have retained copies of mouse chromosomes that contain the 28S ribosomal RNA genes. Images PMID:265531
Mouse homologues of human hereditary disease.
Searle, A G; Edwards, J H; Hall, J G
Details are given of 214 loci known to be associated with human hereditary disease, which have been mapped on both human and mouse chromosomes. Forty two of these have pathological variants in both species; in general the mouse variants are similar in their effects to the corresponding human ones, but exceptions include the Dmd/DMD and Hprt/HPRT mutations which cause little, if any, harm in mice. Possible reasons for phenotypic differences are discussed. In most pathological variants the gene product seems to be absent or greatly reduced in both species. The extensive data on conserved segments between human and mouse chromosomes are used to predict locations in the mouse of over 50 loci of medical interest which are mapped so far only on human chromosomes. In about 80% of these a fairly confident prediction can be made. Some likely homologies between mapped mouse loci and unmapped human ones are also given. Sixty six human and mouse proto-oncogene and growth factor gene homologies are also listed; those of confirmed location are all in known conserved segments. A survey of 18 mapped human disease loci and chromosome regions in which the manifestation or severity of pathological effects is thought to be the result of genomic imprinting shows that most of the homologous regions in the mouse are also associated with imprinting, especially those with homologues on human chromosomes 11p and 15q. Useful methods of accelerating the production of mouse models of human hereditary disease include (1) use of a supermutagen, such as ethylnitrosourea (ENU), (2) targeted mutagenesis involving ES cells, and (3) use of gene transfer techniques, with production of 'knockout mutations'. PMID:8151633
Humanized mouse models in transplantation research.
Hogenes, Marieke; Huibers, Manon; Kroone, Chantal; de Weger, Roel
The interest in the use of humanized mouse models for research topics like Graft versus Host Disease (GvHD), allograft studies and other studies to the human immune system is growing. The design of these models is still improving and enables even more complicated studies to these topics. For researchers it can be difficult to choose the best option from the current pool of available models. The decision will depend on which hypothesis needs to be tested, in which field of interest, and therefore 'the best model' will differ from one to another. In this review, we provide a guide to the most common available humanized mouse models, with regards to different mouse strains, transplantation material, transplantation techniques, pre- and post-conditioning and references to advantages and disadvantages. Also, an evaluation of experiences with humanized mouse models in studies on GvHD and allograft rejection is provided. PMID:24636846
Human homolog of the mouse sperm receptor
Chamberlin, M.E.; Dean, J. (National Institutes of Health, Bethesda, MD (USA))
The human zona pellucida, composed of three glycoproteins (ZP1, ZP2, and ZP3), forms an extracellular matrix that surrounds ovulated eggs and mediates species-specific fertilization. The genes that code for at least two of the zona proteins (ZP2 and ZP3) cross-hybridize with other mammalian DNA. The recently characterized mouse sperm receptor gene (Zp-3) was used to isolate its human homolog. The human homolog spans {approx}18.3 kilobase pairs (kbp) (compared to 8.6 kbp for the mouse gene) and contains eight exons, the sizes of which are strictly conserved between the two species. Four short (8-15 bp) sequences within the first 250 bp of the 5{prime} flanking region in the human Zp-3 homolog are also present upstream of mouse Zp-3. These elements may modulate oocyte-specific gene expression. By using the polymerase chain reaction, a full-length cDNA of human ZP3 was isolated from human ovarian poly(A){sup +} RNA and used to deduce the structure of human ZP3 mRNA. Certain features of the human and mouse ZP3 transcripts are conserved. Both have unusually short 5{prime} and 3{prime} untranslated regions, both contain a single open reading frame that is 74% identical, and both code for 424 amino acid polypeptides that are 67% the same. The similarity between the two proteins may define domains that are important in maintaining the structural integrity of the zona pellucida, while the differences may play a role in mediating the species-specific events of mammalian fertilization.
Human homolog of the mouse sperm receptor.
Chamberlin, M E; Dean, J
The human zona pellucida, composed of three glycoproteins (ZP1, ZP2, and ZP3), forms an extracellular matrix that surrounds ovulated eggs and mediates species-specific fertilization. The genes that code for at least two of the zona proteins (ZP2 and ZP3) cross-hybridize with other mammalian DNA. The recently characterized mouse sperm receptor gene (Zp-3) was used to isolate its human homolog. The human homolog spans approximately 18.3 kilobase pairs (kbp) (compared to 8.6 kbp for the mouse gene) and contains eight exons, the sizes of which are strictly conserved between the two species. Four short (8-15 bp) sequences within the first 250 bp of the 5' flanking region in the human Zp-3 homolog are also present upstream of mouse Zp-3. These elements may modulate oocyte-specific gene expression. By using the polymerase chain reaction, a full-length cDNA of human ZP3 was isolated from human ovarian poly(A)+ RNA and used to deduce the structure of human ZP3 mRNA. Certain features of the human and mouse ZP3 transcripts are conserved. Both have unusually short 5' and 3' untranslated regions, both contain a single open reading frame that is 74% identical, and both code for 424 amino acid polypeptides that are 67% the same. The similarity between the two proteins may define domains that are important in maintaining the structural integrity of the zona pellucida, while the differences may play a role in mediating the species-specific events of mammalian fertilization. Images PMID:2385582
A Humanized Mouse Model of Tuberculosis
Calderon, Veronica E.; Valbuena, Gustavo; Goez, Yenny; Judy, Barbara M.; Huante, Matthew B.; Sutjita, Putri; Johnston, R. Katie; Estes, D. Mark; Hunter, Robert L.; Actor, Jeffrey K.; Cirillo, Jeffrey D.; Endsley, Janice J.
Mycobacterium tuberculosis (M.tb) is the second leading infectious cause of death worldwide and the primary cause of death in people living with HIV/AIDS. There are several excellent animal models employed to study tuberculosis (TB), but many have limitations for reproducing human pathology and none are amenable to the direct study of HIV/M.tb co-infection. The humanized mouse has been increasingly employed to explore HIV infection and other pathogens where animal models are limiting. Our goal was to develop a small animal model of M.tb infection using the bone marrow, liver, thymus (BLT) humanized mouse. NOD-SCID/?cnull mice were engrafted with human fetal liver and thymus tissue, and supplemented with CD34+ fetal liver cells. Excellent reconstitution, as measured by expression of the human CD45 pan leukocyte marker by peripheral blood populations, was observed at 12 weeks after engraftment. Human T cells (CD3, CD4, CD8), as well as natural killer cells and monocyte/macrophages were all observed within the human leukocyte (CD45+) population. Importantly, human T cells were functionally competent as determined by proliferative capacity and effector molecule (e.g. IFN-?, granulysin, perforin) expression in response to positive stimuli. Animals infected intranasally with M.tb had progressive bacterial infection in the lung and dissemination to spleen and liver from 2�8 weeks post infection. Sites of infection in the lung were characterized by the formation of organized granulomatous lesions, caseous necrosis, bronchial obstruction, and crystallization of cholesterol deposits. Human T cells were distributed throughout the lung, liver, and spleen at sites of inflammation and bacterial growth and were organized to the periphery of granulomas. These preliminary results demonstrate the potential to use the humanized mouse as a model of experimental TB. PMID:23691024
Comparative Recombination Rates in the Rat, Mouse, and Human Genomes
Seaman, Michael I.
as much recombination as mouse and rat, and in many mammalian spe- cies females recombine more than males of rat, mouse, and human to estimate local recombination rates across these genomes. Humans have greater overall levels of recombination, as well as greater variance. In rat and mouse, the size of the chromosome
Expression of human and suppression of mouse nucleolus organizer activity in mouse-human somatic cell hybrids.
Miller, O J; Miller, D A; Dev, V G; Tantravahi, R; Croce, C M
Most mouse-human somatic cell hybrids show preferential loss of human chromosomes, absence of human 28S ribosomal RNA, and suppression of human nucleolus organizer activity, as visualized by the Ag-AS silver histochemical stain. In contrast, the mouse-human hybrids studied here show preferential loss of mouse chromosomes. The hybrids were made by fusion of HT-1080-6TG human fibrosarcoma cells with BALB/c mouse peritoneal macrophages or strain 129 mouse teratocarcinoma cells. The Ag-AS staining method shows nucleolus organizer activity of chromosomes 13, 14, 15, 21 (rarely), and 22 in the human parent and chromosomes 12, 15, 16 (rarely), and 18 in the BALB/c mouse parent. In the hybrid cells the human nucleolus organizer regions are active, as shown by Ag-AS staining and involvement in "satellite association." The mouse nucleolus organizer regions are not stained by the Ag-AS method even though mouse chromosomes 12, 15, and 18 are present in the BALB/c hybrids and at least one copy of each mouse chromosome is present in the teratocarcinoma-derived hybrids. Thus, in these mouse-human hybrids, unlike those that lose human chromosomes, only human nucleolus organizer activity is expressed, and mouse nucleolus organizer activity is suppressed. Images PMID:1070003
A Comparison of Senescence in Mouse and Human Cells
Vera Gorbunova; Andrei Seluanov
\\u000a Senescence is observed in both human and mouse cells, however, there are fundamental differences in how senescence is controlled\\u000a between the two species. Human fibroblasts undergo replicative senescence as a result of telomere shortening. In contrast,\\u000a mouse fibroblasts do not senesce when grown at a physiological oxygen concentration. In atmospheric oxygen, mouse cells enter\\u000a a state that resembles senescence, but
The Mouse Genome Database (MGD): facilitating mouse as a model for human biology and disease.
Eppig, Janan T; Blake, Judith A; Bult, Carol J; Kadin, James A; Richardson, Joel E
The Mouse Genome Database (MGD, http://www.informatics.jax.org) serves the international biomedical research community as the central resource for integrated genomic, genetic and biological data on the laboratory mouse. To facilitate use of mouse as a model in translational studies, MGD maintains a core of high-quality curated data and integrates experimentally and computationally generated data sets. MGD maintains a unified catalog of genes and genome features, including functional RNAs, QTL and phenotypic loci. MGD curates and provides functional and phenotype annotations for mouse genes using the Gene Ontology and Mammalian Phenotype Ontology. MGD integrates phenotype data and associates mouse genotypes to human diseases, providing critical mouse-human relationships and access to repositories holding mouse models. MGD is the authoritative source of nomenclature for genes, genome features, alleles and strains following guidelines of the International Committee on Standardized Genetic Nomenclature for Mice. A new addition to MGD, the Human-Mouse: Disease Connection, allows users to explore gene-phenotype-disease relationships between human and mouse. MGD has also updated search paradigms for phenotypic allele attributes, incorporated incidental mutation data, added a module for display and exploration of genes and microRNA interactions and adopted the JBrowse genome browser. MGD resources are freely available to the scientific community. PMID:25348401
The Mouse Genome Database (MGD): facilitating mouse as a model for human biology and disease
Eppig, Janan T.; Blake, Judith A.; Bult, Carol J.; Kadin, James A.; Richardson, Joel E.
The Mouse Genome Database (MGD, http://www.informatics.jax.org) serves the international biomedical research community as the central resource for integrated genomic, genetic and biological data on the laboratory mouse. To facilitate use of mouse as a model in translational studies, MGD maintains a core of high-quality curated data and integrates experimentally and computationally generated data sets. MGD maintains a unified catalog of genes and genome features, including functional RNAs, QTL and phenotypic loci. MGD curates and provides functional and phenotype annotations for mouse genes using the Gene Ontology and Mammalian Phenotype Ontology. MGD integrates phenotype data and associates mouse genotypes to human diseases, providing critical mouse�human relationships and access to repositories holding mouse models. MGD is the authoritative source of nomenclature for genes, genome features, alleles and strains following guidelines of the International Committee on Standardized Genetic Nomenclature for Mice. A new addition to MGD, the Human�Mouse: Disease Connection, allows users to explore gene�phenotype�disease relationships between human and mouse. MGD has also updated search paradigms for phenotypic allele attributes, incorporated incidental mutation data, added a module for display and exploration of genes and microRNA interactions and adopted the JBrowse genome browser. MGD resources are freely available to the scientific community. PMID:25348401
Comparative anatomy of mouse and human nail units.
Fleckman, Philip; Jaeger, Karin; Silva, Kathleen A; Sundberg, John P
Recent studies of mice with hair defects have resulted in major contributions to the understanding of hair disorders. To use mouse models as a tool to study nail diseases, a basic understanding of the similarities and differences between the human and mouse nail unit is required. In this study we compare the human and mouse nail unit at the macroscopic and microscopic level and use immunohistochemistry to determine the keratin expression patterns in the mouse nail unit. Both species have a proximal nail fold, cuticle, nail matrix, nail bed, nail plate, and hyponychium. Distinguishing features are the shape of the nail and the presence of an extended hyponychium in the mouse. Expression patterns of most keratins are similar. These findings indicate that the mouse nail unit shares major characteristics with the human nail unit and overall represents a very similar structure, useful for the investigation of nail diseases and nail biology. PMID:23408541
A Detailed Comparison of Mouse and Human Cardiac Development
Krishnan, Anita; Samtani, Rajeev; Dhanantwari, Preeta; Lee, Elaine; Yamada, Shigehito; Shiota, Kohei; Donofrio, Mary T.; Leatherbury, Linda; Lo, Cecilia W.
Background Mouse mutants are used to model human congenital cardiovascular disease. Little is published comparing normal cardiovascular development in mice versus humans. We carried out a systematic comparative analysis of mouse and human fetal cardiovascular development. Methods Episcopic fluorescence image capture (EFIC) was performed on 66 wild type mouse embryos from embryonic day (E) 9.5-birth; 2D and 3D datasets were compared with EFIC and magnetic resonance images (MRI) from a study of 52 human fetuses (Carnegie Stage (CS) 13�23). Results Time course of atrial, ventricular and outflow septation were outlined, and followed a similar sequence in both species. Bilateral vena cavae and prominent atrial appendages were seen in the mouse fetus; in human fetuses, atrial appendages were small, and a single right superior vena cava was present. In contrast to humans with separate pulmonary vein orifices, a pulmonary venous confluence with one orifice enters the left atrium in mice. Conclusions The cardiac developmental sequences observed in mouse and human fetuses are comparable, with minor differences in atrial and venous morphology. These comparisons of mouse and human cardiac development strongly support that mouse morphogenesis is a good model for human development. PMID:25167202
Genomic responses in mouse models poorly mimic human inflammatory diseases
Seok, Junhee; Warren, H. Shaw; Cuenca, Alex G.; Mindrinos, Michael N.; Baker, Henry V.; Xu, Weihong; Richards, Daniel R.; McDonald-Smith, Grace P.; Gao, Hong; Hennessy, Laura; Finnerty, Celeste C.; L�pez, Cecilia M.; Honari, Shari; Moore, Ernest E.; Minei, Joseph P.; Cuschieri, Joseph; Bankey, Paul E.; Johnson, Jeffrey L.; Sperry, Jason; Nathens, Avery B.; Billiar, Timothy R.; West, Michael A.; Jeschke, Marc G.; Klein, Matthew B.; Gamelli, Richard L.; Gibran, Nicole S.; Brownstein, Bernard H.; Miller-Graziano, Carol; Calvano, Steve E.; Mason, Philip H.; Cobb, J. Perren; Rahme, Laurence G.; Lowry, Stephen F.; Maier, Ronald V.; Moldawer, Lyle L.; Herndon, David N.; Davis, Ronald W.; Xiao, Wenzhong; Tompkins, Ronald G.; Abouhamze, Amer; Balis, Ulysses G. J.; Camp, David G.; De, Asit K.; Harbrecht, Brian G.; Hayden, Douglas L.; Kaushal, Amit; O�Keefe, Grant E.; Kotz, Kenneth T.; Qian, Weijun; Schoenfeld, David A.; Shapiro, Michael B.; Silver, Geoffrey M.; Smith, Richard D.; Storey, John D.; Tibshirani, Robert; Toner, Mehmet; Wilhelmy, Julie; Wispelwey, Bram; Wong, Wing H
A cornerstone of modern biomedical research is the use of mouse models to explore basic pathophysiological mechanisms, evaluate new therapeutic approaches, and make go or no-go decisions to carry new drug candidates forward into clinical trials. Systematic studies evaluating how well murine models mimic human inflammatory diseases are nonexistent. Here, we show that, although acute inflammatory stresses from different etiologies result in highly similar genomic responses in humans, the responses in corresponding mouse models correlate poorly with the human conditions and also, one another. Among genes changed significantly in humans, the murine orthologs are close to random in matching their human counterparts (e.g., R2 between 0.0 and 0.1). In addition to improvements in the current animal model systems, our study supports higher priority for translational medical research to focus on the more complex human conditions rather than relying on mouse models to study human inflammatory diseases. PMID:23401516
An analysis on gene architecture in human and mouse genomes.
Sakharkar, Meena Kishore; Perumal, Bagavathi S; Sakharkar, Kishore R; Kangueane, Pandjassarame
A comparative genome analysis on exon-intron distribution profiles is performed for human and mouse genomes to deduce similarities and differences between them. Interestingly, both in human and mouse genomes, the total length in introns and intergenic DNA on each chromosome is significantly correlated to the chromosome size. The results presented provide a framework for understanding the nature and patterns of exon-intron length distributions, the constraints on them and their role in genome design and evolution. PMID:16268780
Activation of human complement by mouse and mouse/human chimeric monoclonal antibodies.
Seino, J; Eveleigh, P; Warnaar, S; van Haarlem, L J; van Es, L A; Daha, M R
The complement (C)-activating capabilities in human serum of 32 mouse and 10 mouse/human chimeric MoAbs of different isotypes, and their fragments, were tested in vitro. Activation of C via the classical pathway (CP) was performed in 1% factor D-deficient serum in gelatin containing Veronal buffer in the presence of calcium and magnesium (GVB++), while activation of the alternative pathway of C (AP) was assessed in 10% C1q-depleted serum in the presence of 5 mM MgCl2 in GVB++. The C-activating ability of MoAbs was expressed relative to the degree of activation of complement by aggregated IgG for the CP and relative to mouse IgG1 for the AP. All of seven mouse IgG2a MoAbs were potent activators of the CP. The results of CP activation by IgG1, IgG2b and IgG3 isotypes were different for individual MoAbs. Only three (two IgG1 and one IgG3) of 32 mouse MoAbs were potent activators of the AP. IgG2a and IgG2b were relatively poor AP activators. There were a few MoAbs which activated both the AP and CP. Of 10 chimeric MoAbs, two IgG1, one IgG2 and one IgG4 were poor or non-activators of the CP. On the other hand, IgG2 and IgG4 were good AP activators. IgG3 was the most potent AP activator. Most of the F(ab')2 fragments were activators of the AP and displayed no activation of the CP. Fc fragments only activated the CP, whereas Fab' did not activate the CP or the AP. These studies suggest that the route of complement activation by class and subclass MoAbs can not always be predicted in advance and based only on their subclass identity. PMID:8222320
Cell Host & Microbe A Mouse Model for the Human Pathogen
Galan, Jorge E
infection. A S. Typhi mutant in a gene required for virulence in humans was unable to replicateCell Host & Microbe Resource A Mouse Model for the Human Pathogen Salmonella Typhi Jeongmin Song,1.09.003 SUMMARY Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever, a life-threatening human
ORIGINAL PAPER A transgenic mouse model engineered to investigate human
Devignes, Marie-Dominique
ORIGINAL PAPER A transgenic mouse model engineered to investigate human brain-derived neurotrophic to study human BDNF gene expression and permit the screening of compounds capable of stimulating its activity. A 145-kb yeast artificial chromosome carrying the human BDNF gene has been engi- neered
Stepwise Development of MAIT Cells in Mouse and Human
Paris-Sud XI, Universit� de
Stepwise Development of MAIT Cells in Mouse and Human Emmanuel Martin1,2[ , Emmanuel Treiner1%�4% of blood T cells. Finally, in contrast with NKT cells, human MAIT cell development is independent the ZBTB16 transcription factor, which, in contrast, is expressed by NKT cells and the memory human MAIT
Cytoarchitecture of mouse and rat cingulate cortex with human homologies.
Vogt, Brent A; Paxinos, George
A gulf exists between cingulate area designations in human neurocytology and those used in rodent brain atlases with a major underpinning of the former being midcingulate cortex (MCC). The present study used images extracted from the Franklin and Paxinos mouse atlas and Paxinos and Watson rat atlas to demonstrate areas comprising MCC and modifications of anterior cingulate (ACC) and retrosplenial cortices. The laminar architecture not available in the atlases is also provided for each cingulate area. Both mouse and rat have a MCC with neurons in all layers that are larger than in ACC and layer Va has particularly prominent neurons and reduced neuron densities. An undifferentiated ACC area 33 lies along the rostral callosal sulcus in rat but not in mouse and area 32 has dorsal and ventral subdivisions with the former having particularly large pyramidal neurons in layer Vb. Both mouse and rat have anterior and posterior divisions of retrosplenial areas 29c and 30, although their cytology is different in rat and mouse. Maps of the rodent cingulate cortices provide for direct comparisons with each region in the human including MCC and it is significant that rodents do not have a posterior cingulate region composed of areas 23 and 31 like the human. It is concluded that rodents and primates, including humans, possess a MCC and this homology along with those in ACC and retrosplenial cortices permit scientists inspired by human considerations to test hypotheses on rodent models of human diseases. PMID:23229151
Mouse models of acute, chemical itch and pain in humans
LaMotte, Robert H.; Shimada, Steven G.; Sikand, Parul
In psychophysical experiments, humans use different verbal responses to pruritic and algesic chemical stimuli to indicate the different qualities of sensation they feel. A major challenge for behavioral models in the mouse of chemical itch and pain in humans is to devise experimental protocols that provide the opportunity for the animal to exhibit a multiplicity of responses as well. One basic criterion is that chemicals that evoke primarily itch or pain in humans should elicit different types of responses when applied in the same way to the mouse. Meeting this criterion is complicated by the fact that the type of behavioral responses exhibited by the mouse depends in part on the site of chemical application such as the nape of the neck which evokes only scratching with the hind paw vs. the hind limb which elicits licking and biting. Here, we review to what extent mice behaviorally differentiate chemicals that elicit itch vs. pain in humans. PMID:21929688
The relevance of genetically altered mouse models of human disease.
Bhogal, Nirmala; Combes, Robert
The impetus to develop useful models of human disease and toxicity has resulted in a number of large-scale mouse mutagenesis programmes. This, in turn, has stimulated considerable concern regarding the scientific validity and welfare of genetically altered mice, and the large numbers of mice that are required by such programmes. In this paper, the scientific advantages and limitations of genetically altered mice as models of several human diseases are discussed. We conclude that, while the use of some such mouse models has contributed considerably to an understanding of human disease and toxicity, other genetically altered mouse models have limited scientific relevance, and fewer have positively contributed to the development of novel human medicines. Suggestions for improving this unsatisfactory situation are made. PMID:16945009
End Sequencing and Finger Printing of Human & Mouse BAC Libraries
Fraser, C.
This project provided for continued end sequencing of existing and new BAC libraries constructed to support human sequencing as well as to initiate BAC end sequencing from the mouse BAC libraries constructed to support mouse sequencing. The clones, the sequences, and the fingerprints are now an available resource for the community at large. Research and development of new metaodologies for BAC end sequencing have reduced costs and increase throughput.
Mouse Model for Human Arginase Deficiency
Ramaswamy K. Iyer; Paul K. Yoo; Rita M. Kern; Nora Rozengurt; Rosemarie Tsoa; William E. O'Brien; Hong Yu; Wayne W. Grody; Stephen D. Cederbaum
Deficiency of liver arginase (AI) causes hyperargininemia (OMIM 207800), a disorder characterized by progressive mental impairment, growth retardation, and spasticity and punctuated by sometimes fatal epi- sodes of hyperammonemia. We constructed a knockout mouse strain carrying a nonfunctional AI gene by homologous recombination. Arginase AI knockout mice completely lacked liver arginase (AI) activity, exhibited severe symptoms of hyperammonemia, and died
Further characterization of the genetic defect of the Bent tail mouse, a mouse model for human neural tube defects
E. D. Klootwijk; Mascha M. V. A. P. Schijvenaars; Edwin C. M. Mariman; Barbara Franke
BACKGROUND: Neural tube defects (NTDs) are congenital malformations arising mostly from incomplete neural tube closure during early embryogenesis. Most NTDs in humans have a complex etiology, with involvement of both genetic and environmental factors. More than 100 mouse models for human neural tube defects exist; Bent tail is one of them. The mouse mutant is caused by a submicroscopic deletion
Liver immune-pathogenesis and therapy of human liver tropic virus infection in humanized mouse models
Bility, Moses T; Li, Feng; Cheng, Liang; Su, Lishan
Hepatitis B virus (HBV) and hepatitis C virus (HCV) infect and replicate primarily in human hepatocytes. Few reliable and easy accessible animal models are available for studying the immune system�s contribution to the liver disease progression during hepatitis virus infection. Humanized mouse models reconstituted with human hematopoietic stem cells (HSCs) have been developed to study human immunology, human immunodeficiency virus 1 infection, and immunopathogenesis. However, a humanized mouse model engrafted with both human immune and human liver cells is needed to study infection and immunopathogenesis of HBV/HCV infection in vivo. We have recently developed the humanized mouse model with both human immune and human liver cells (AFC8-hu HSC/Hep) to study immunopathogenesis and therapy of HCV infection in vivo. In this review, we summarize the current models of HBV/HCV infection and their limitations in immunopathogenesis. We will then present our recent findings of HCV infection and immunopathogenesis in the AFC8-hu HSC/Hep mouse, which supports HCV infection, human T-cell response and associated liver pathogenesis. Inoculation of humanized mice with primary HCV isolates resulted in long-term HCV infection. HCV infection induced elevated infiltration of human immune cells in the livers of HCV-infected humanized mice. HCV infection also induced HCV-specific T-cell immune response in lymphoid tissues of humanized mice. Additionally, HCV infection induced liver fibrosis in humanized mice. Anti-human alpha smooth muscle actin (?SMA) staining showed elevated human hepatic stellate cell activation in HCV-infected humanized mice. We discuss the limitation and future improvements of the AFC8-hu HSC/Hep mouse model and its application in evaluating novel therapeutics, as well as studying both HCV and HBV infection, human immune responses, and associated human liver fibrosis and cancer. PMID:23855307
A comparison of the mouse and human lipoproteome: suitability of the mouse model for studies of human lipoproteins.
Gordon, Scott M; Li, Hailong; Zhu, Xiaoting; Shah, Amy S; Lu, L Jason; Davidson, W Sean
Plasma levels of low density lipoproteins (LDL) and high density lipoproteins (HDL) exhibit opposing associations with cardiovascular disease in human populations and mouse models have been heavily used to derive a mechanistic understanding of these relationships. In humans, recent mass spectrometry studies have revealed that the plasma lipoproteome is significantly more complex than originally appreciated. This is particularly true for HDL which contains some 90 distinct proteins, a majority of which play functional roles that go beyond those expected for simple lipid transport. Unfortunately, the mouse lipoproteome remains largely uncharacterized-a significant gap given the heavy reliance on the model. Using a gel filtration chromatography and mass spectrometry analysis that targets phospholipid-bound plasma proteins, we compared the mouse lipoproteome and its size distribution to a previous, identical human analysis. We identified 113 lipid associated proteins in the mouse. In general, the protein diversity in the LDL and HDL size ranges was similar in mice versus humans, though some distinct differences were noted. For the majority of proteins, the size distributions, that is, whether a given protein was associated with large versus small HDL particles, for example, were also similar between species. Again, however, there were clear differences exhibited by a minority of proteins that may reflect metabolic differences between species. Finally, by correlating the lipid and protein size profiles, we identified five proteins that closely track with the major HDL protein, apolipoprotein A-I across both species. Thus, mice have most of the minor proteins identified in human lipoproteins that play key roles in inflammation, innate immunity, proteolysis and its inhibition, and vitamin transport. This provides support for the continued use of the mouse as a model for many aspects of human lipoprotein metabolism. PMID:25894274
Zicam-Induced Damage to Mouse and Human Nasal Tissue
Lim, Jae H.; Davis, Greg E.; Wang, Zhenshan; Li, Vicky; Wu, Yuping; Rue, Tessa C.; Storm, Daniel R.
Intranasal medications are used to treat various nasal disorders. However, their effects on olfaction remain unknown. Zicam (zinc gluconate; Matrixx Initiatives, Inc), a homeopathic substance marketed to alleviate cold symptoms, has been implicated in olfactory dysfunction. Here, we investigated Zicam and several common intranasal agents for their effects on olfactory function. Zicam was the only substance that showed significant cytotoxicity in both mouse and human nasal tissue. Specifically, Zicam-treated mice had disrupted sensitivity of olfactory sensory neurons to odorant stimulation and were unable to detect novel odorants in behavioral testing. These findings were long-term as no recovery of function was observed after two months. Finally, human nasal explants treated with Zicam displayed significantly elevated extracellular lactate dehydrogenase levels compared to saline-treated controls, suggesting severe necrosis that was confirmed on histology. Our results demonstrate that Zicam use could irreversibly damage mouse and human nasal tissue and may lead to significant smell dysfunction. PMID:19876403
Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature
Melican, Keira; Aubey, Flore; Dum�nil, Guillaume
Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream. PMID:24747976
A fully humanized transgenic mouse model of Huntington disease
Southwell, Amber L.; Warby, Simon C.; Carroll, Jeffrey B.; Doty, Crystal N.; Skotte, Niels H.; Zhang, Weining; Villanueva, Erika B.; Kovalik, Vlad; Xie, Yuanyun; Pouladi, Mahmoud A.; Collins, Jennifer A.; Yang, X. William; Franciosi, Sonia; Hayden, Michael R.
Silencing the mutant huntingtin gene (muHTT) is a direct and simple therapeutic strategy for the treatment of Huntington disease (HD) in principle. However, targeting the HD mutation presents challenges because it is an expansion of a common genetic element (a CAG tract) that is found throughout the genome. Moreover, the HTT protein is important for neuronal health throughout life, and silencing strategies that also reduce the wild-type HTT allele may not be well tolerated during the long-term treatment of HD. Several HTT silencing strategies are in development that target genetic sites in HTT that are outside of the CAG expansion, including HD mutation-linked single-nucleotide polymorphisms and the HTT promoter. Preclinical testing of these genetic therapies has required the development of a new mouse model of HD that carries these human-specific genetic targets. To generate a fully humanized mouse model of HD, we have cross-bred BACHD and YAC18 on the Hdh?/? background. The resulting line, Hu97/18, is the first murine model of HD that fully genetically recapitulates human HD having two human HTT genes, no mouse Hdh genes and heterozygosity of the HD mutation. We find that Hu97/18 mice display many of the behavioral changes associated with HD including motor, psychiatric and cognitive deficits, as well as canonical neuropathological abnormalities. This mouse line will be useful for gaining additional insights into the disease mechanisms of HD as well as for testing genetic therapies targeting human HTT. PMID:23001568
Genome-Wide Expression Profiling of Five Mouse Models Identifies Similarities and Differences with Human Psoriasis
William R. Swindell; Andrew Johnston; Steve Carbajal; Gangwen Han; Christian Wohn; Jun Lu; Xianying Xing; Rajan P. Nair; John J. Voorhees; James T. Elder; Xiao-Jing Wang; Shigetoshi Sano; Errol P. Prens; John Digiovanni; Mark R. Pittelkow; Nicole L. Ward; Johann E. Gudjonsson; Stefan Bereswill
Development of a suitable mouse model would facilitate the investigation of pathomechanisms underlying human psoriasis and would also assist in development of therapeutic treatments.However, while many psoriasis mouse models have been proposed, no single model recapitulates all features of the human disease, and standardized validation criteria for psoriasis mouse models have not been widely applied. In this study, whole-genome transcriptional
Differences between human and mouse embryonic stem cells
Irene Ginis; Yongquan Luo; Takumi Miura; Scott Thies; Ralph Brandenberger; Sharon Gerecht-Nir; Michal Amit; Ahmet Hoke; Melissa K Carpenter; Joseph Itskovitz-Eldor; Mahendra S Rao
We compared gene expression profiles of mouse and human ES cells by immunocytochemistry, RT-PCR, and membrane-based focused cDNA array analysis. Several markers that in concert could distinguish undifferentiated ES cells from their differentiated progeny were identified. These included known markers such as SSEA antigens, OCT3\\/4, SOX-2, REX-1 and TERT, as well as additional markers such as UTF-1, TRF1, TRF2, connexin43,
STING activator c-di-GMP enhances the anti-tumor effects of peptide vaccines in melanoma-bearing mice.
Wang, Zili; Celis, Esteban
Therapeutic vaccines to induce anti-tumor CD8 T cells have been used in clinical trials for advanced melanoma patients, but the clinical response rate and overall survival time have not improved much. We believe that these dismal outcomes are caused by inadequate number of antigen-specific CD8 T cells generated by most vaccines. In contrast, huge CD8 T cell responses readily occur during acute viral infections. High levels of type-I interferon (IFN-I) are produced during these infections, and this cytokine not only exhibits anti-viral activity but also promotes CD8 T cell responses. The studies described here were performed to determine whether promoting the production of IFN-I could enhance the potency of a peptide vaccine. We report that cyclic diguanylate monophosphate (c-di-GMP), which activates the stimulator of interferon genes, potentiated the immunogenicity and anti-tumor effects of a peptide vaccine against mouse B16 melanoma. The synergistic effects of c-di-GMP required co-administration of costimulatory anti-CD40 antibody, the adjuvant poly-IC, and were mediated in part by IFN-I. These findings demonstrate that peptides representing CD8 T cell epitopes can be effective inducers of large CD8 T cell responses in vaccination strategies that mimic acute viral infections. PMID:25986168
A novel antiangiogenic and vascular normalization therapy targeted against human CD160 receptor
Chabot, Sophie; Bigot, Karine; Tabiasco, Julie; Provost, Alexandra; Golzio, Muriel; Noman, Muhammad Zaeem; Giustiniani, J�r�me; Bellard, Elisabeth; Brayer, St�phanie; Aguerre-Girr, Maryse; Meggetto, Fabienne; Giuriato, Sylvie; Malecaze, Fran�ois; Galiacy, St�phane; Ja�s, Jean-Philippe; Chose, Olivier; Kadouche, Jean; Chouaib, Salem; Teissi�, Justin; Abitbol, Marc; Bensussan, Armand
Angiogenesis plays an essential role in several diseases of the eye and in the growth of solid tumors, but existing antiangiogenic therapies have limited benefits in several cases. We report the antiangiogenic effects of a monoclonal antibody, CL1-R2, in several animal models of neovascularization. CL1-R2 recognizes human CD160, a membrane receptor which is conserved in various mammal species. We show that CD160 is expressed on the endothelial cells of newly formed blood vessels in human colon carcinoma and mouse B16 melanoma but not in vessels of healthy tissues. CL1-R2 reduced fibroblast growth factor 2�induced neovascularization in the rabbit cornea, in a mouse model of oxygen-induced retinopathy, and in a mouse Matrigel plug assay. Treatment of B16 melanoma-bearing mice with CL1-R2 combined with cyclophosphamide chemotherapy caused regression of the tumor vasculature and normalization of the remaining vessels as shown by Doppler ultrasonography, intravital microscopy, and histology. These studies validate CD160 as a potential new target in cases of human pathological ocular and tumor neoangiogenesis that do not respond or become resistant to existing antiangiogenic drugs. PMID:21482699
From XenoMouse technology to panitumumab, the first fully human antibody product from transgenic mice
Rafael G Amado; Xiaodong Yang; Lorin Roskos; Gisela Schwab; Aya Jakobovits
Therapeutic monoclonal antibodies have shown limited efficacy and safety owing to immunogenicity of mouse sequences in humans. Among the approaches developed to overcome these hurdles were transgenic mice genetically engineered with a 'humanized' humoral immune system. One such transgenic system, the XenoMouse, has succeeded in recapitulating the human antibody response in mice, by introducing nearly the entire human immunoglobulin loci
Further Improvements of the P. falciparum Humanized Mouse Model
Meija, Pedro; Swetman, Claire; Gleeson, James; P�rignon, Jean-Louis; Druilhe, Pierre
Background It has been shown previously that it is possible to obtain growth of Plasmodium falciparum in human erythrocytes grafted in mice lacking adaptive immune responses by controlling, to a certain extent, innate defences with liposomes containing clodronate (clo-lip). However, the reproducibility of those models is limited, with only a proportion of animals supporting longstanding parasitemia, due to strong inflammation induced by P. falciparum. Optimisation of the model is much needed for the study of new anti-malarial drugs, drug combinations, and candidate vaccines. Materials/Methods We investigated the possibility of improving previous models by employing the intravenous route (IV) for delivery of both human erythrocytes (huRBC) and P. falciparum, instead of the intraperitoneal route (IP), by testing various immunosuppressive drugs that might help to control innate mouse defences, and by exploring the potential benefits of using immunodeficient mice with additional genetic defects, such as those with IL-2R? deficiency (NSG mice). Results We demonstrate here the role of aging, of inosine and of the IL-2 receptor ? mutation in controlling P. falciparum induced inflammation. IV delivery of huRBC and P. falciparum in clo-lip treated NSG mice led to successful infection in 100% of inoculated mice, rapid rise of parasitemia to high levels (up to 40%), long-lasting parasitemia, and consistent results from mouse-to-mouse. Characteristics were closer to human infection than in previous models, with evidence of synchronisation, partial sequestration, and receptivity to various P. falciparum strains without preliminary adaptation. However, results show that a major IL-12p70 inflammatory response remains prevalent. Conclusion The combination of the NSG mouse, clodronate loaded liposomes, and IV delivery of huRBC has produced a reliable and more relevant model that better meets the needs of Malaria research. PMID:21483851
A simple methodology for conversion of mouse monoclonal antibody to human-mouse chimeric form.
Dang, Vinh T; Mandakhalikar, Kedar D; Ng, Oi-Wing; Tan, Yee-Joo
Passive immunotherapy has mainly been used as a therapy against cancer and inflammatory conditions. Recent studies have shown that monoclonal antibody-(mAb-) based passive immunotherapy is a promising approach to combat virus infection. Specific mouse mAbs can be routinely generated in large amounts with the use of hybridoma technology but these cannot be used for therapy in human beings due to their immunogenicity. Therefore, the development of chimeric and humanized mAbs is important for therapeutic purpose. This is facilitated by a variety of molecular techniques like recombinant DNA technology and the better understanding of the structure and function of antibody. The human-mouse chimeric forms allow detailed analysis of the mechanism of inhibition and the potential for therapeutic applications. Here, a step-by-step description of the conversion process will be described. The commercial availability of the reagents required in each step means that this experimentation can be easily set up in research laboratories. PMID:24078817
CYP1A1 and CYP1A2 expression: Comparing 'humanized' mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines
Uno, Shigeyuki; Endo, Kaori [Department of Biochemistry, Nihon University School of Medicine, 30-1 Oyaguchikami-cho, Itabashi-ku, Tokyo 173-8610 (Japan); Ishida, Yuji; Tateno, Chise [PhenixBio Co., Ltd., 3-4-1 Kagamiyama, Higashihiroshima, Hiroshima 739-0046 (Japan); Makishima, Makoto [Department of Biochemistry, Nihon University School of Medicine, 30-1 Oyaguchikami-cho, Itabashi-ku, Tokyo 173-8610 (Japan); Yoshizato, Katsutoshi [PhenixBio Co., Ltd., 3-4-1 Kagamiyama, Higashihiroshima, Hiroshima 739-0046 (Japan); Nebert, Daniel W. [Department of Environmental Health and Center for Environmental Genetics (CEG) University of Cincinnati Medical Center, P.O. Box 670056, Cincinnati OH 45267-0056 (United States)], E-mail: email@example.com
Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how 'human-like' can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1{sub C}YP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carrying humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+){sub s}evere-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs.
Mouse liver repopulation with hepatocytes generated from human fibroblasts
Zhu, Saiyong; Rezvani, Milad; Harbell, Jack; Mattis, Aras N.; Wolfe, Alan R.; Benet, Leslie Z.; Willenbring, Holger; Ding, Sheng
Human induced pluripotent stem cells (iPSCs) promise to revolutionize research and therapy of liver diseases by providing a source of hepatocytes for autologous cell therapy and disease modeling. However, despite progress in advancing the differentiation of iPSCs into hepatocytes (iPSC-Heps) in vitro1�3, cells that replicate the ability of human primary adult hepatocytes (aHeps) to proliferate extensively in vivo have not been reported. This deficiency has hampered efforts to recreate human liver diseases in mice, and has cast doubt on the potential of iPSC-Heps for liver cell therapy. The reason is that extensive post-transplant expansion is needed to establish and sustain a therapeutically effective liver cell mass in patients, a lesson learned from clinical trials of aHep transplantation4. As a solution to this problem, we report generation of human fibroblast-derived hepatocytes that can repopulate mouse livers. Unlike current protocols for deriving hepatocytes from human fibroblasts, ours did not generate iPSCs, but shortcut reprogramming to pluripotency to generate an induced multipotent progenitor cell (iMPC) state from which endoderm progenitor cells (iMPC-EPCs) and subsequently hepatocytes (iMPC-Heps) could be efficiently differentiated. For this, we identified small molecules that aided endoderm and hepatocyte differentiation without compromising proliferation. After transplantation into an immune-deficient mouse model of human liver failure, iMPC-Heps proliferated extensively and acquired levels of hepatocyte function similar to aHeps. Unfractionated iMPC-Heps did not form tumors, most likely because they never entered a pluripotent state. To our knowledge, this is the first demonstration of significant liver repopulation of mice with human hepatocytes generated in vitro, which removes a long-standing roadblock on the path to autologous liver cell therapy. PMID:24572354
Mouse liver repopulation with hepatocytes generated from human fibroblasts.
Zhu, Saiyong; Rezvani, Milad; Harbell, Jack; Mattis, Aras N; Wolfe, Alan R; Benet, Leslie Z; Willenbring, Holger; Ding, Sheng
Human induced pluripotent stem cells (iPSCs) have the capability of revolutionizing research and therapy of liver diseases by providing a source of hepatocytes for autologous cell therapy and disease modelling. However, despite progress in advancing the differentiation of iPSCs into hepatocytes (iPSC-Heps) in vitro, cells that replicate the ability of human primary adult hepatocytes (aHeps) to proliferate extensively in vivo have not been reported. This deficiency has hampered efforts to recreate human liver diseases in mice, and has cast doubt on the potential of iPSC-Heps for liver cell therapy. The reason is that extensive post-transplant expansion is needed to establish and sustain a therapeutically effective liver cell mass in patients, a lesson learned from clinical trials of aHep transplantation. Here, as a solution to this problem, we report the generation of human fibroblast-derived hepatocytes that can repopulate mouse livers. Unlike current protocols for deriving hepatocytes from human fibroblasts, ours did not generate iPSCs but cut short reprogramming to pluripotency to generate an induced multipotent progenitor cell (iMPC) state from which endoderm progenitor cells and subsequently hepatocytes (iMPC-Heps) could be efficiently differentiated. For this purpose we identified small molecules that aided endoderm and hepatocyte differentiation without compromising proliferation. After transplantation into an immune-deficient mouse model of human liver failure, iMPC-Heps proliferated extensively and acquired levels of hepatocyte function similar to those of aHeps. Unfractionated iMPC-Heps did not form tumours, most probably because they never entered a pluripotent state. Our results establish the feasibility of significant liver repopulation of mice with human hepatocytes generated in vitro, which removes a long-standing roadblock on the path to autologous liver cell therapy. PMID:24572354
This review focuses on the relationships between mouse skin tumors and human lung cancer and discusses these relationships from several perspectives. hese perspectives include: mouse skin as an experimental test system; metabolic comparisons of the response of mouse skin and huma...
Binding of licarbazepine enantiomers to mouse and human plasma proteins.
Fortuna, Ana; Alves, Gilberto; Falc�o, Am�lcar; Soares-da-Silva, Patr�cio
Racemic licarbazepine (Lic) is the active metabolite of oxcarbazepine (OXC) and eslicarbazepine acetate (ESL), appearing in human plasma as S-licarbazepine (S-Lic) and R-licarbazepine (R-Lic). However, human metabolism of OXC and ESL to Lic differs in the S-Lic/R-Lic enantiomeric ratio observed in plasma. S-Lic appears in higher proportion after ESL administration than after OXC (95% versus 80%). Enantioselective pharmacokinetics of Lic enantiomers have been found in mice after their separate administration and in humans following OXC treatment. Since protein binding of drugs may be enantioselective and a determining factor of pharmacokinetics, the binding of S-Lic and R-Lic to mouse and human total plasma proteins and, specifically, to human serum albumin (HSA) and alpha(1)-acid glycoprotein (AGP) were herein investigated for the first time. Free and bound fractions of S-Lic and R-Lic were separated by ultrafiltration after previous in vitro incubation of spiked plasma samples and protein solutions with each enantiomer at 10, 25 and 50 microg/ml. The results revealed that the extent of binding of Lic enantiomers to total plasma proteins was 30% and independent of the drug concentration and species considered. The data also suggest that the binding of Lic enantiomers to HSA is greater than that to AGP. Moreover, absence of enantioselectivity in the binding of Lic enantiomers to mouse and human plasma proteins and to HSA and AGP is evident. In conclusion, these findings suggest that the enantioselectivity observed in vivo in the biodisposition of S-Lic and R-Lic is not dependent on their affinity to plasma proteins. PMID:20578208
Gene Expression and Functional Annotation of the Human and Mouse Choroid Plexus Epithelium
Janssen, Sarah F.; van der Spek, Sophie J. F.; ten Brink, Jacoline B.; Essing, Anke H. W.; Gorgels, Theo G. M. F.; van der Spek, Peter J.; Jansonius, Nomdo M.; Bergen, Arthur A. B.
Background The choroid plexus epithelium (CPE) is a lobed neuro-epithelial structure that forms the outer blood-brain barrier. The CPE protrudes into the brain ventricles and produces the cerebrospinal fluid (CSF), which is crucial for brain homeostasis. Malfunction of the CPE is possibly implicated in disorders like Alzheimer disease, hydrocephalus or glaucoma. To study human genetic diseases and potential new therapies, mouse models are widely used. This requires a detailed knowledge of similarities and differences in gene expression and functional annotation between the species. The aim of this study is to analyze and compare gene expression and functional annotation of healthy human and mouse CPE. Methods We performed 44k Agilent microarray hybridizations with RNA derived from laser dissected healthy human and mouse CPE cells. We functionally annotated and compared the gene expression data of human and mouse CPE using the knowledge database Ingenuity. We searched for common and species specific gene expression patterns and function between human and mouse CPE. We also made a comparison with previously published CPE human and mouse gene expression data. Results Overall, the human and mouse CPE transcriptomes are very similar. Their major functionalities included epithelial junctions, transport, energy production, neuro-endocrine signaling, as well as immunological, neurological and hematological functions and disorders. The mouse CPE presented two additional functions not found in the human CPE: carbohydrate metabolism and a more extensive list of (neural) developmental functions. We found three genes specifically expressed in the mouse CPE compared to human CPE, being ACE, PON1 and TRIM3 and no human specifically expressed CPE genes compared to mouse CPE. Conclusion Human and mouse CPE transcriptomes are very similar, and display many common functionalities. Nonetheless, we also identified a few genes and pathways which suggest that the CPE between mouse and man differ with respect to transport and metabolic functions. PMID:24391755
An In Vivo Mouse Model of Metastatic Human Thyroid Cancer
Zhang, Lisa; Gaskins, Kelli; Yu, Zhiya; Xiong, Yin; Merino, Maria J.
Background: Mouse models of metastatic human cancers are important tools in preclinical studies for testing new systematic therapies and studying effectors of cancer metastasis. The major drawbacks of current mouse models for metastatic thyroid cancer are that they have low metastasis rates and do not allow in vivo tumor detection. Here, we report and characterize an in vivo detectable metastasis mouse model of human thyroid cancer using multiple thyroid cancer cell lines. Methods: Human anaplastic thyroid cancer cell lines 8505C, C-643, SW-1736, and THJ-16T; follicular thyroid cancer cell lines FTC-133, FTC-236, and FTC-238; and H�rthle cell carcinoma cell line XTC-1 were transfected with a linearized pGL4.51[luc2/CMV/Neo] vector or transduced with lentivirus containing Luc2-eGFP reporter genes. The stably transfected cells were injected intravenously into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice. Tumors were detected with an in vivo imaging system�Xenogen IVIS. Vemurafenib, a BRAF inhibitor, was used to treat lung metastases generated from 8505C-Luc2 cells with a BRAFV600E mutation to test the accuracy of the model to evaluate response to therapy. Results: Intravenous injection of as few as 30,000 8505C-Luc2 cells produced lung metastases in 100% of the injected mice, and many of these mice also developed bone metastases at a later stage of the disease. Similarly, metastatic tumors also developed in all mice injected with C-643-Luc2, THJ-16T-Luc2, FTC-133-Luc2, FTC-236-Luc2, FTC-238-Luc2, and XTC-1-Luc2 cells. The metastases were easily detectable in vivo, and tumor progression could be dynamically and accurately followed and correlated with the actual tumor burden. Furthermore, disease progression could be easily controlled by adjusting the number of injected cells. The in vivo treatment of 8505C xenograft lung metastases with vemurafenib dramatically reduced the growth and signal intensity with good correlation with actual tumor burden. Conclusions: Herein we report an in vivo detectable mouse model of metastatic human thyroid cancer that is reliable and reproducible. It will serve as a useful tool in the preclinical testing of alternative systematic therapies for metastatic thyroid cancer, and for functional studies of thyroid cancer tumor biology in vivo. PMID:24262022
Glycine receptor mouse mutants: model systems for human hyperekplexia
Schaefer, Natascha; Langlhofer, Georg; Kluck, Christoph J; Villmann, Carmen
Human hyperekplexia is a neuromotor disorder caused by disturbances in inhibitory glycine-mediated neurotransmission. Mutations in genes encoding for glycine receptor subunits or associated proteins, such as GLRA1, GLRB, GPHN and ARHGEF9, have been detected in patients suffering from hyperekplexia. Classical symptoms are exaggerated startle attacks upon unexpected acoustic or tactile stimuli, massive tremor, loss of postural control during startle and apnoea. Usually patients are treated with clonazepam, this helps to dampen the severe symptoms most probably by up-regulating GABAergic responses. However, the mechanism is not completely understood. Similar neuromotor phenotypes have been observed in mouse models that carry glycine receptor mutations. These mouse models serve as excellent tools for analysing the underlying pathomechanisms. Yet, studies in mutant mice looking for postsynaptic compensation of glycinergic dysfunction via an up-regulation in GABAA receptor numbers have failed, as expression levels were similar to those in wild-type mice. However, presynaptic adaptation mechanisms with an unusual switch from mixed GABA/glycinergic to GABAergic presynaptic terminals have been observed. Whether this presynaptic adaptation explains the improvement in symptoms or other compensation mechanisms exist is still under investigation. With the help of spontaneous glycine receptor mouse mutants, knock-in and knock-out studies, it is possible to associate behavioural changes with pharmacological differences in glycinergic inhibition. This review focuses on the structural and functional characteristics of the various mouse models used to elucidate the underlying signal transduction pathways and adaptation processes and describes a novel route that uses gene-therapeutic modulation of mutated receptors to overcome loss of function mutations. PMID:23941355
Human-mouse comparative genomics: successes and failures to reveal functional regions of the human genome
Pennacchio, Len A.; Baroukh, Nadine; Rubin, Edward M.
Deciphering the genetic code embedded within the human genome remains a significant challenge despite the human genome consortium's recent success at defining its linear sequence (Lander et al. 2001; Venter et al. 2001). While useful strategies exist to identify a large percentage of protein encoding regions, efforts to accurately define functional sequences in the remaining {approx}97 percent of the genome lag. Our primary interest has been to utilize the evolutionary relationship and the universal nature of genomic sequence information in vertebrates to reveal functional elements in the human genome. This has been achieved through the combined use of vertebrate comparative genomics to pinpoint highly conserved sequences as candidates for biological activity and transgenic mouse studies to address the functionality of defined human DNA fragments. Accordingly, we describe strategies and insights into functional sequences in the human genome through the use of comparative genomics coupled wit h functional studies in the mouse.
Principles Of Regulatory Information Conservation Between Mouse And Human
Kim, Bong-Hyun; Wu, Weisheng; Cayting, Philip; Boyle, Alan P.; Sundaram, Vasavi; Xing, Xiaoyun; Dogan, Nergiz; Li, Jingjing; Euskirchen, Ghia; Lin, Shin; Lin, Yiing; Visel, Axel; Kawli, Trupti; Yang, Xinqiong; Patacsil, Dorrelyn; Keller, Cheryl A.; Giardine, Belinda; Kundaje, Anshul; Wang, Ting; Pennacchio, Len A.; Weng, Zhiping; Hardison, Ross C.; Snyder, Michael P.
Summary To broaden our understanding of the evolution of gene regulation mechanisms, we generated occupancy profiles for 34 orthologous transcription factors (TFs) in human-mouse erythroid progenitor, lymphoblast, and embryonic stem cell lines. By combining the genome-wide TF occupancy repertoires, associated epigenetic signals, and TF co-association patterns, we deduced several evolutionary principles of gene regulatory features operating since the mouse and human lineages diverged. The genomic distribution profiles, primary binding motifs, chromatin states, and DNA methylation preferences are well conserved for TF occupied sequences (TF OSs). However, the extent to which orthologous DNA segments are bound by orthologous TFs varies both among TFs and with genomic location: binding at promoters is more highly conserved than binding at distal elements. Importantly, occupancy conserved TF OSs tend to be pleiotropic; they function in multiple tissues and also co-associate with multiple TFs. Single nucleotide variants (SNVs) at sites with potential regulatory functions are enriched in occupancy conserved TF OSs. PMID:25409826
Immunohistochemical assessment of parafibromin in mouse and human tissues
Porzionato, Andrea; Macchi, Veronica; Barzon, Luisa; Masi, Giulia; Iacobone, Maurizio; Parenti, Anna; Pal�, Giorgio; De Caro, Raffaele
Parafibromin is a protein encoded by the HRPT2 oncosuppressor gene, whose mutation causes the hyperparathyroidism�jaw tumour syndrome, characterized by the occurrence of parathyroid adenoma or carcinoma, fibro-osseous jaw tumours, and renal neoplastic and non-neoplastic abnormalities. Non-morphological techniques, such as Northern and Western blotting and reverse transcriptase-PCR, indicate that parafibromin is ubiquitously expressed, but extensive immunohistochemical studies have not been performed. To increase our knowledge of the distribution and patterns of expression of parafibromin, we examined its expression and location in many different mouse and human organs by immunohistochemistry. There were no substantial differences in parafibromin expression between mouse and human. We found widespread expression of parafibromin, except in connective tissue, smooth muscle, endothelium and some other types of epithelia (colonic, urinary, tubaric, uterine, thyroid). Heterogeneity of positivity intensity and subcellular location (nuclear, nucleocytoplasmic, cytoplasmic) was found between tissues and cell types, suggesting differential functional involvement of parafibromin. Moreover, higher parafibromin expression was found in cell types, such as hepatocytes, cells of the base of gastric glands, renal cortex tubules and the pars intermedia of the hypophysis, which are characterized by different proliferative capacity, thus indicating that the cellular function of parafibromin may not be reduced only to its anti-proliferative effect. PMID:17118068
A Comparison of Some Organizational Characteristics of the Mouse Central Retina and the Human Macula
Hoo, Juyea; Yee, Claudine; Williams, David S.
Mouse models have greatly assisted our understanding of retinal degenerations. However, the mouse retina does not have a macula, leading to the question of whether the mouse is a relevant model for macular degeneration. In the present study, a quantitative comparison between the organization of the central mouse retina and the human macula was made, focusing on some structural characteristics that have been suggested to be important in predisposing the macula to stresses leading to degeneration: photoreceptor density, phagocytic load on the RPE, and the relative thinness of Bruch�s membrane. Light and electron microscopy measurements from retinas of two strains of mice, together with published data on human retinas, were used for calculations and subsequent comparisons. As in the human retina, the central region of the mouse retina possesses a higher photoreceptor cell density and a thinner Bruch�s membrane than in the periphery; however, the magnitudes of these periphery to center gradients are larger in the human. Of potentially greater relevance is the actual photoreceptor cell density, which is much greater in the mouse central retina than in the human macula, underlying a higher phagocytic load for the mouse RPE. Moreover, at eccentricities that correspond to the peripheral half of the human macula, the rod to cone ratio is similar between mouse and human. Hence, with respect to photoreceptor density and phagocytic load of the RPE, the central mouse retina models at least the more peripheral part of the macula, where macular degeneration is often first evident. PMID:25923208
Update of the human and mouse SERPIN gene superfamily
The serpin family comprises a structurally similar, yet functionally diverse, set of proteins. Named originally for their function as serine proteinase inhibitors, many of its members are not inhibitors but rather chaperones, involved in storage, transport, and other roles. Serpins are found in genomes of all kingdoms, with 36 human protein-coding genes and five pseudogenes. The mouse has 60 Serpin functional genes, many of which are orthologous to human SERPIN genes and some of which have expanded into multiple paralogous genes. Serpins are found in tissues throughout the body; whereas most are extracellular, there is a class of intracellular serpins. Serpins appear to have roles in inflammation, immune function, tumorigenesis, blood clotting, dementia, and cancer metastasis. Further characterization of these proteins will likely reveal potential biomarkers and therapeutic targets for disease. PMID:24172014
A short-term assay utilizing a human/mouse monochromosomal hybrid cell line R3-5, to detect chemically induced aneuploidy in mammalian cells is described. A single human chromosome transferred into mouse cells was used as a cytogenetic marker to quantitate abnormal chromosome seg...
Human embryonic stem cells with biological and epigenetic to those of mouse ESCs
Jaenisch, Rudolf
Human and mouse embryonic stem cells (ESCs) are derived from blastocyst-stage embryos but have very different biological properties, and molecular analyses suggest that the pluripotent state of human ESCs isolated so far ...
Sequence homologies and linkage group conservation of the human and mouse Cenpc genes
McKay, S.; Thomson, E.; Cooke, H. [Western General Hospital, Edinburgh (United Kingdom)] [Western General Hospital, Edinburgh (United Kingdom)
Using a previously identified human CENPC cDNA fragment, the authors have isolated cDNA clones corresponding to the complete mouse Cenpc coding sequence. Using these cDNAs as probes to genomic libraries, the authors have isolated genomic clones corresponding to the mouse and human genes and also to a mouse pseudogene. In situ hybridization mapping of these genes reveals that the human gene maps to 4q12-q13.3 and the mouse gene to 5E2-E5. These sites are in a region of linkage group conservation between the two species. Secondary sites are present in man on chromosome 12q21.2-q21.33 and in mouse on chromosome 2B. This mouse secondary site is a pseudogene on the basis of DNA sequence. These secondary sites are not syntenic in the two species. 25 refs., 2 figs., 1 tab.
Altered glucose metabolism in mouse and humans conceived by IVF.
Chen, Miaoxin; Wu, Linda; Zhao, Junli; Wu, Fang; Davies, Michael J; Wittert, Gary A; Norman, Robert J; Robker, Rebecca L; Heilbronn, Leonie K
In vitro fertilization (IVF) may influence the metabolic health of children. However, in humans, it is difficult to separate out the relative contributions of genetics, environment, or the process of IVF, which includes ovarian stimulation (OS) and embryo culture. Therefore, we examined glucose metabolism in young adult humans and in adult male C57BL/6J mice conceived by IVF versus natural birth under energy-balanced and high-fat-overfeeding conditions. In humans, peripheral insulin sensitivity, as assessed by hyperinsulinemic-euglycemic clamp (80 mU/m(2)/min), was lower in IVF patients (n = 14) versus control subjects (n = 20) after 3 days of an energy-balanced diet (30% fat). In response to 3 days of overfeeding (+1,250 kcal/day, 45% fat), there was a greater increase in systolic blood pressure in IVF versus controls (P = 0.02). Mice conceived after either OS alone or IVF weighed significantly less at birth versus controls (P < 0.01). However, only mice conceived by IVF displayed increased fasting glucose levels, impaired glucose tolerance, and reduced insulin-stimulated Akt phosphorylation in the liver after 8 weeks of consuming either a chow or high-fat diet (60% fat). Thus, OS impaired fetal growth in the mouse, but only embryo culture resulted in changes in glucose metabolism that may increase the risk of the development of metabolic diseases later in life, in both mice and humans. PMID:24760136
Human but Not Mouse Hepatocytes Respond to Interferon-Lambda In Vivo
Hermant, Pascale; Demarez, C�line; Mahlak�iv, Tanel; Staeheli, Peter; Meuleman, Philip; Michiels, Thomas
The type III interferon (IFN) receptor is preferentially expressed by epithelial cells. It is made of two subunits: IFNLR1, which is specific to IFN-lambda (IFN-?) and IL10RB, which is shared by other cytokine receptors. Human hepatocytes express IFNLR1 and respond to IFN-?. In contrast, the IFN-? response of the mouse liver is very weak and IFNLR1 expression is hardly detectable in this organ. Here we investigated the IFN-? response at the cellular level in the mouse liver and we tested whether human and mouse hepatocytes truly differ in responsiveness to IFN-?. When monitoring expression of the IFN-responsive Mx genes by immunohistofluorescence, we observed that the IFN-? response in mouse livers was restricted to cholangiocytes, which form the bile ducts, and that mouse hepatocytes were indeed not responsive to IFN-?. The lack of mouse hepatocyte response to IFN-? was observed in different experimental settings, including the infection with a hepatotropic strain of influenza A virus which triggered a strong local production of IFN-?. With the help of chimeric mice containing transplanted human hepatocytes, we show that hepatocytes of human origin readily responded to IFN-? in a murine environment. Thus, our data suggest that human but not mouse hepatocytes are responsive to IFN-? in vivo. The non-responsiveness is an intrinsic property of mouse hepatocytes and is not due to the mouse liver micro-environment. PMID:24498220
Diffusion of (2-/sup 14/C)diazepam across hairless mouse skin and human skin
Koch, R.L.; Palicharla, P.; Groves, M.J.
The objectives of this study were to investigate the absorption of diazepam applied topically to the hairless mouse in vivo and to determine the diffusion of diazepam across isolated hairless mouse skin and human skin. (/sup 14/C)Diazepam was readily absorbed after topical administration to the intact hairless mouse, a total of 75.8% of the /sup 14/C-label applied being recovered in urine and feces. Diazepam was found to diffuse across human and hairless mouse skin unchanged in experiments with twin-chambered diffusion cells. The variation in diffusion rate or the flux for both human and mouse tissues was greater among specimens than between duplicate or triplicate trials for a single specimen. Fluxes for mouse skin (stratum corneum, epidermis, and dermis) were greater than for human skin (stratum corneum and epidermis): 0.35-0.61 microgram/cm2/h for mouse skin vs 0.24-0.42 microgram/cm2/h for human skin. The permeability coefficients for mouse skin ranged from 1.4-2.4 X 10(-2)cm/h compared with 0.8-1.4 X 10(-2)cm/h for human skin. Although human stratum corneum is almost twice the thickness of that of the hairless mouse, the diffusion coefficients for human skin were 3-12 times greater (0.76-3.31 X 10(-6) cm2/h for human skin vs 0.12-0.27 X 10(-6) cm2/h for hairless mouse) because of a shorter lag time for diffusion across human skin. These differences between the diffusion coefficients and diffusion rates (or permeability coefficients) suggest that the presence of the dermis may present some barrier properties. In vitro the dermis may require complete saturation before the diazepam can be detected in the receiving chamber.
The top skin-associated genes: a comparative analysis of human and mouse skin transcriptomes.
Gerber, Peter Arne; Buhren, Bettina Alexandra; Schrumpf, Holger; Homey, Bernhard; Zlotnik, Albert; Hevezi, Peter
The mouse represents a key model system for the study of the physiology and biochemistry of skin. Comparison of skin between mouse and human is critical for interpretation and application of data from mouse experiments to human disease. Here, we review the current knowledge on structure and immunology of mouse and human skin. Moreover, we present a systematic comparison of human and mouse skin transcriptomes. To this end, we have recently used a genome-wide database of human gene expression to identify genes highly expressed in skin, with no, or limited expression elsewhere - human skin-associated genes (hSAGs). Analysis of our set of hSAGs allowed us to generate a comprehensive molecular characterization of healthy human skin. Here, we used a similar database to generate a list of mouse skin-associated genes (mSAGs). A comparative analysis between the top human (n=666) and mouse (n=873) skin-associated genes (SAGs) revealed a total of only 30.2% identity between the two lists. The majority of shared genes encode proteins that participate in structural and barrier functions. Analysis of the top functional annotation terms revealed an overlap for morphogenesis, cell adhesion, structure, and signal transduction. The results of this analysis, discussed in the context of published data, illustrate the diversity between the molecular make up of skin of both species and grants a probable explanation, why results generated in murine in vivo models often fail to translate into the human. PMID:24497224
From XenoMouse technology to panitumumab, the first fully human antibody product from transgenic mice.
Jakobovits, Aya; Amado, Rafael G; Yang, Xiaodong; Roskos, Lorin; Schwab, Gisela
Therapeutic monoclonal antibodies have shown limited efficacy and safety owing to immunogenicity of mouse sequences in humans. Among the approaches developed to overcome these hurdles were transgenic mice genetically engineered with a 'humanized' humoral immune system. One such transgenic system, the XenoMouse, has succeeded in recapitulating the human antibody response in mice, by introducing nearly the entire human immunoglobulin loci into the germ line of mice with inactivated mouse antibody machinery. XenoMouse strains have been used to generate numerous high-affinity, fully human antibodies to targets in multiple disease indications, many of which are progressing in clinical development. However, validation of the technology has awaited the recent regulatory approval of panitumumab (Vectibix), a fully human antibody directed against epidermal growth factor receptor (EGFR), as treatment for people with advanced colorectal cancer. The successful development of panitumumab represents a milestone for mice engineered with a human humoral immune system and their future applications. PMID:17921999
Fibrillin genes map to regions of conserved mouse/human synteny on mouse chromosomes 2 and 18.
Li, X; Pereira, L; Zhang, H; Sanguineti, C; Ramirez, F; Bonadio, J; Francke, U
Fibrillin proteins are major structural components of the 10-nm microfibrils found in elastic and nonelastic connective tissues. Previous studies have mapped the human genes for two fibrillins to chromosome bands 15q21 (FBN1) and 5q23-q31 (FBN2) and have demonstrated that FBN1 mutations are associated with Marfan syndrome, while FBN2 is linked to the gene for congenital contractural arachnodactyly. Here, we report the isolation of genomic clones of the corresponding mouse fibrillin genes (Fbn-1 and Fbn-2). By analyzing a mapping panel of mouse x rodent somatic hybrid cell lines, we have assigned the Fbn-1 gene to mouse chromosome 2 and the Fbn-2 gene to mouse chromosome 18. We then sublocalized the fibrillin genes to bands 2F (Fbn-1) and 18D-E1 (Fbn-2) by fluorescence in situ hybridization. These regions are known to exhibit conserved synteny with the regions on human chromosomes 15 and 5 that carry the homologous human fibrillin genes. In addition, the Fbn-1 gene maps in the vicinity of the gene for a connective tissue disorder on mouse chromosome 2 called Tight-skin (Tsk). PMID:8307578
Unique forms of human and mouse nuclear receptor corepressor SMRT
Ordentlich, Peter; Downes, Michael; Xie, Wen; Genin, Anna; Spinner, Nancy B.; Evans, Ronald M.
Nuclear hormone receptors have been shown to repress transcription in the absence of ligand. This repression is mediated by a corepressor complex that contains the Sin3A protein and histone deacetylases (HDAC1 and 2). Studies by several groups demonstrate that this complex is recruited to nuclear receptors through the highly related corepressors SMRT (silencing mediator of retinoid acid and thyroid hormone receptor) and N-CoR (nuclear receptor corepressor). We describe here the cloning, characterization, and chromosomal mapping of forms of human and mouse SMRT that includes a 1,000-aa extension, which reveals striking homology to the amino terminus of N-CoR. Structure and function studies of wild-type and natural splicing variants suggest the presence of 3�4 amino terminal domains that repress in a cooperative as well as mechanistically distinct fashion. PMID:10077563
A novel DNA sequence motif in human and mouse genomes
Zhang, Shilu; Du, Fang; Ji, Hongkai
We report a novel DNA sequence motif in human and mouse genomes. This motif has several interesting features indicating that it is highly likely to be an unknown functional sequence element. The motif is highly enriched in promoter regions. Locations of the motif sites in the genome have strong tendency to be clustered together. Motif sites are associated with increased phylogenetic conservation as well as elevated DNase I hypersensitivity (DHS) in ENCODE cell lines. Clustered motif sites are found in promoter regions of a substantial fraction of the protein-coding genes in the genome. All together, these indicate that the motif may have important functions associated with a large number of genes. PMID:25990515
A Transgenic Mouse Model for Human Autosomal Dominant Cataract
Hsu, Cheng-Da; Kymes, Steven; Petrash, J. Mark
Purpose To characterize lenses from transgenic mice designed to express mutant and wild-type ?A-crystallin subunits. Methods A series of transgenic mouse strains was created to express mutant (R116C) and wild-type human ?A-crystallin in fiber cells of the lens. Dissected lenses were phenotypically scored for the presence and extent of opacities, fiber cell morphology, and posterior suture morphology. Gene transcripts derived from integrated transgenes were detected by reverse transcriptase-PCR. Distribution of expressed transgenic protein was determined by immunohistochemical staining of lens tissue sections. The abundance of endogenous and transgenic lens proteins was estimated by quantitative Western blot analysis. Results Expression of R116C mutant ?A-crystallin subunits resulted in posterior cortical cataracts and abnormalities associated with the posterior suture. The severity of lens abnormalities did not increase between the ages of 9 and 30 weeks. With respect to opacities and morphologic abnormalities, lenses from transgenic mice that express wild-type human ?A-crystallin subunits were indistinguishable from age-matched non-transgenic control mice. Similar phenotypes were observed in different independent lines of R116C transgenic mice that differed by at least two orders of magnitude in the expression level of the mutant transgenic protein. Conclusions The results show that lens opacities and posterior sutural defects occur when mutant R116C ?A-crystallin subunits are expressed on the background of wild-type endogenous mouse ?-crystallins. Low levels of R116C ?A-crystallin subunits are sufficient to induce lens opacities and sutural defects. PMID:16639013
Detailed comparative map of human chromosome 19q and related regions of the mouse genome
Stubbs, L.; Shannon, M.E.; Kim, Joomyeong [Oak Ridge National Lab., TN (United States)] [and others] [Oak Ridge National Lab., TN (United States); and others
One of the larger contiguous blocks of mouse-human genomic homology includes the proximal portion of mouse chromosome 7 and the long arm of human chromosome 19. Previous studies have demonstrated the close relationship between the two regions, but have also indicated significant rearrangements in the relative orders of homologous mouse and human genes. Here we present the genetic locations of the homologs of 42 human chromosome 19q markers in the mouse, with an emphasis on genes also included in the human chromosome 19 physical map. Our results demonstrate that despite an overall inversion of sequences relative to the centromere, apparent {open_quotes}transpositions{close_quotes} of three gene-rich segments, and a local inversion of markers mapping near the 19q telomere, gene content, order, and spacing are remarkably well conserved throughout the lengths of these related mouse and humans regions. Although most human 19q markers have remained genetically linked in mouse, one small human segment forms a separate region of homology between human chromosome 19q and mouse chromosome 17. Three of the four rearrangements of mouse versus human 19q sequences involve segments that are located directly adjacent to each other in 19q13.3-q13.4, suggesting either the coincident occurrence of these events or their common association with unstable DNA sequences. These data permit an unusually in-depth examination of this large region of mouse-human genomic homology and provide an important new tool to aid in the mapping of genes and associated phenotypes in both species. 66 refs., 3 figs., 1 tab.
A family of candidate taste receptors in human and mouse.
Matsunami, H; Montmayeur, J P; Buck, L B
The gustatory system of mammals can sense four basic taste qualities, bitter, sweet, salty and sour, as well as umami, the taste of glutamate. Previous studies suggested that the detection of bitter and sweet tastants by taste receptor cells in the mouth is likely to involve G-protein-coupled receptors. Although two putative G-protein-coupled bitter/sweet taste receptors have been identified, the chemical diversity of bitter and sweet compounds leads one to expect that there is a larger number of different receptors. Here we report the identification of a family of candidate taste receptors (the TRBs) that are members of the G-protein-coupled receptor superfamily and that are specifically expressed by taste receptor cells. A cluster of genes encoding human TRBs is located adjacent to a Prp gene locus, which in mouse is tightly linked to the SOA genetic locus that is involved in detecting the bitter compound sucrose octaacetate. Another TRB gene is found on a human contig assigned to chromosome 5p15, the location of a genetic locus (PROP) that controls the detection of the bitter compound 6-n-propyl-2-thiouracil in humans. PMID:10766242
NSDL National Science Digital Library
Based in New York City, the MOUSE organization works to empower "underserved students to provide technology support and leadership in their schools, supporting their academic and career success." On their homepage, visitors can learn about their programs, learn about supporting the MOUSE organization, and read up on their resources. In the "Resources" area, visitors can learn about their outreach activities in New York City, Chicago, and California. Visitors working in educational outreach will appreciate the information offered here, including materials on how different groups can receive assistance from the MOUSE organization. Also, visitors can look over the "News" updates to learn about their new programs, their educational seminars, and their outreach activities.
Identification and analysis of alternative splicing events conserved in human and mouse
Yeo, Gene W.; Van Nostrand, Eric; Holste, Dirk; Poggio, Tomaso; Burge, Christopher B.
Alternative pre-mRNA splicing affects a majority of human genes and plays important roles in development and disease. Alternative splicing (AS) events conserved since the divergence of human and mouse are likely of primary biological importance, but relatively few of such events are known. Here we describe sequence features that distinguish exons subject to evolutionarily conserved AS, which we call alternative conserved exons (ACEs), from other orthologous human/mouse exons and integrate these features into an exon classification algorithm, acescan. Genome-wide analysis of annotated orthologous human�mouse exon pairs identified ?2,000 predicted ACEs. Alternative splicing was verified in both human and mouse tissues by using an RT-PCR-sequencing protocol for 21 of 30 (70%) predicted ACEs tested, supporting the validity of a majority of acescan predictions. By contrast, AS was observed in mouse tissues for only 2 of 15 (13%) tested exons that had EST or cDNA evidence of AS in human but were not predicted ACEs, and AS was never observed for 11 negative control exons in human or mouse tissues. Predicted ACEs were much more likely to preserve the reading frame and less likely to disrupt protein domains than other AS events and were enriched in genes expressed in the brain and in genes involved in transcriptional regulation, RNA processing, and development. Our results also imply that the vast majority of AS events represented in the human EST database are not conserved in mouse. PMID:15708978
The utilization of humanized mouse models for the study of human retroviral infections
Van Duyne, Rachel; Pedati, Caitlin; Guendel, Irene; Carpio, Lawrence; Kehn-Hall, Kylene; Saifuddin, Mohammed; Kashanchi, Fatah
The development of novel techniques and systems to study human infectious diseases in both an in vitro and in vivo settings is always in high demand. Ideally, small animal models are the most efficient method of studying human afflictions. This is especially evident in the study of the human retroviruses, HIV-1 and HTLV-1, in that current simian animal models, though robust, are often expensive and difficult to maintain. Over the past two decades, the construction of humanized animal models through the transplantation and engraftment of human tissues or progenitor cells into immunocompromised mouse strains has allowed for the development of a reconstituted human tissue scaffold in a small animal system. The utilization of small animal models for retroviral studies required expansion of the early CB-17 scid/scid mouse resulting in animals demonstrating improved engraftment efficiency and infectivity. The implantation of uneducated human immune cells and associated tissue provided the basis for the SCID-hu Thy/Liv and hu-PBL-SCID models. Engraftment efficiency of these tissues was further improved through the integration of the non-obese diabetic (NOD) mutation leading to the creation of NODSCID, NOD/Shi-scid IL2r?-/-, and NOD/SCID ?2-microglobulinnull animals. Further efforts at minimizing the response of the innate murine immune system produced the Rag2-/-?c-/- model which marked an important advancement in the use of human CD34+ hematopoietic stem cells. Together, these animal models have revolutionized the investigation of retroviral infections in vivo. PMID:19674458
Bone metastasis in a novel breast cancer mouse model containing human breast and human bone.
Xia, Tian-Song; Wang, Guo-Zhu; Ding, Qiang; Liu, Xiao-An; Zhou, Wen-Bin; Zhang, Yi-Fen; Zha, Xiao-Ming; Du, Qing; Ni, Xiao-Jian; Wang, Jue; Miao, Su-Yu; Wang, Shui
In practice, investigations for bone metastasis of breast cancer rely heavily on models in vivo. Lacking of such ideal model makes it difficult to study the whole process or accurate mechanism of each step of this metastatic disease. Development of xenograft mouse models has made great contributions in this area. Currently, the best animal model of breast cancer metastasizing to bone is NOD/SCID-hu models containing human bone, which makes it possible to let the breast cancer cells and the bone target of osteotropic metastasis be both of human origin. We have developed a novel mouse model containing both human bone and breast, and proved it functional and reliable. In this study, a set of human breast cancer cell line including MDA-MB-231, MDA-MB-231BO, MCF-7, ZR-75-1 and SUM1315 were characterized their osteotropism in this model. A specific cell line SUM1315 made species-specific bone metastasis, certifying the osteotropism-identification utility of the novel mouse model. Furthermore, gene expression and microRNA expression profiling analysis were done to the two SUM1315 derived sub lines isolated and purified from the orthotopic and metastatic xenograft. In addition, to demonstrate the disparity between the "spontaneous" and "forced" bone metastasis in mouse model, MDA-MB-231 cells were inoculated into both the human implants in this model simultaneously, and then primary cultured and profiling analyzed. Supported by overall results of profiling analyses, this study suggested the novel model was a useful tool for understanding, preventing and treating bone metastasis of breast cancer, meanwhile it had provided significant information for further investigations. PMID:21638054
Diffusion of [2-14C]Diazepam Across Hairless Mouse Skin and Human Skin
Ronald L. Koch; Prema Palicharla; Michael J. Groves
The objectives of this study were to investigate the absorption of diazepam applied topically to the hairless mouse in vivo and to determine the diffusion of diazepam across isolated hairless mouse skin and human skin. [14C]Diazepam was readily absorbed after topical administration to the intact hairless mouse, a total of 75.8% of the 14C-label applied being recovered in urine and
Continuous delivery of human and mouse erythropoietin in mice by genetically engineered polymer encapsulated myoblasts
E R�gulier; BL Schneider; N D�glon; Y Beuzard; P Aebischer
The transplantation of polymer encapsulated myoblasts genetically engineered to secrete erythropoietin (Epo) may obviate the need for repeated parenteral administration of recombinant Epo as a treatment for chronic renal failure, cancer or AIDS-associated anemia. To explore this possibility, the human and mouse Epo cDNAs under the control of the housekeeping mouse PGK-1 promoter were transfected into mouse C2C12 myoblasts, which
Genomic cloning of mouse MIF (macrophage inhibitory factor) and genetic mapping of the human and mouse expressed gene and nine mouse pseudogenes
Kozak, C.A.; Adamson, M.C.; Buckler, C.E. [National Institute of Allergy and Infectious Diseases, Bethesda, MD (United States)] [and others] [National Institute of Allergy and Infectious Diseases, Bethesda, MD (United States); and others
The single functional mouse gene for MIF (macrophage migration inhibitory factor) has been cloned from a P1 library, and its exon/intron structure determined and shown to resemble that of the human gene. The gene was mapped to chromosome 10 using two multilocus crosses between laboratory strains and either Mus musculus or Mus spretus. Nine additional loci containing related sequences, apparently all processed pseudogenes, were also mapped to chromosomes 1, 2, 3, 7, 8, 9, 12, 17, and 19. While most of these pseudogenes were also found in inbred mice and M. spretus, some are species specific. This suggests that there have been active phases of pseudogene formation in Mus both before and after the separation of musculus and spretus. The human gene contains no pseudogene; we assigned the human gene to chromosome 19, consistent with the location of mouse and human functional genes for MIF in a region of conserved linkage. 43 refs., 4 figs., 1 tab.
Recommended nomenclature for five mammalian carboxylesterase gene families: human, mouse, and rat genes and proteins.
Holmes, Roger S; Wright, Matthew W; Laulederkind, Stanley J F; Cox, Laura A; Hosokawa, Masakiyo; Imai, Teruko; Ishibashi, Shun; Lehner, Richard; Miyazaki, Masao; Perkins, Everett J; Potter, Phillip M; Redinbo, Matthew R; Robert, Jacques; Satoh, Tetsuo; Yamashita, Tetsuro; Yan, Bingfan; Yokoi, Tsuyoshi; Zechner, Rudolf; Maltais, Lois J
Mammalian carboxylesterase (CES or Ces) genes encode enzymes that participate in xenobiotic, drug, and lipid metabolism in the body and are members of at least five gene families. Tandem duplications have added more genes for some families, particularly for mouse and rat genomes, which has caused confusion in naming rodent Ces genes. This article describes a new nomenclature system for human, mouse, and rat carboxylesterase genes that identifies homolog gene families and allocates a unique name for each gene. The guidelines of human, mouse, and rat gene nomenclature committees were followed and "CES" (human) and "Ces" (mouse and rat) root symbols were used followed by the family number (e.g., human CES1). Where multiple genes were identified for a family or where a clash occurred with an existing gene name, a letter was added (e.g., human CES4A; mouse and rat Ces1a) that reflected gene relatedness among rodent species (e.g., mouse and rat Ces1a). Pseudogenes were named by adding "P" and a number to the human gene name (e.g., human CES1P1) or by using a new letter followed by ps for mouse and rat Ces pseudogenes (e.g., Ces2d-ps). Gene transcript isoforms were named by adding the GenBank accession ID to the gene symbol (e.g., human CES1_AB119995 or mouse Ces1e_BC019208). This nomenclature improves our understanding of human, mouse, and rat CES/Ces gene families and facilitates research into the structure, function, and evolution of these gene families. It also serves as a model for naming CES genes from other mammalian species. PMID:20931200
Divergence of human and mouse brain transcriptome highlights Alzheimer disease pathways
Miller, Jeremy A.; Horvath, Steve; Geschwind, Daniel H.
Because mouse models play a crucial role in biomedical research related to the human nervous system, understanding the similarities and differences between mouse and human brain is of fundamental importance. Studies comparing transcription in human and mouse have come to varied conclusions, in part because of their relatively small sample sizes or underpowered methodologies. To better characterize gene expression differences between mouse and human, we took a systems-biology approach by using weighted gene coexpression network analysis on more than 1,000 microarrays from brain. We find that global network properties of the brain transcriptome are highly preserved between species. Furthermore, all modules of highly coexpressed genes identified in mouse were identified in human, with those related to conserved cellular functions showing the strongest between-species preservation. Modules corresponding to glial and neuronal cells were sufficiently preserved between mouse and human to permit identification of cross species cell-class marker genes. We also identify several robust human-specific modules, including one strongly correlated with measures of Alzheimer disease progression across multiple data sets, whose hubs are poorly-characterized genes likely involved in Alzheimer disease. We present multiple lines of evidence suggesting links between neurodegenerative disease and glial cell types in human, including human-specific correlation of presenilin-1 with oligodendrocyte markers, and significant enrichment for known neurodegenerative disease genes in microglial modules. Together, this work identifies convergent and divergent pathways in mouse and human, and provides a systematic framework that will be useful for understanding the applicability of mouse models for human brain disorders. PMID:20616000
Retinoic acid has different effects on UCP1 expression in mouse and human adipocytes
Background Increased adipose thermogenesis is being considered as a strategy aimed at preventing or reversing obesity. Thus, regulation of the uncoupling protein 1 (UCP1) gene in human adipocytes is of significant interest. Retinoic acid (RA), the carboxylic acid form of vitamin A, displays agonist activity toward several nuclear hormone receptors, including RA receptors (RARs) and peroxisome proliferator-activated receptor ? (PPAR?). Moreover, RA is a potent positive regulator of UCP1 expression in mouse adipocytes. Results The effects of all-trans RA (ATRA) on UCP1 gene expression in models of mouse and human adipocyte differentiation were investigated. ATRA induced UCP1 expression in all mouse white and brown adipocytes, but inhibited or had no effect on UCP1 expression in human adipocyte cell lines and primary human white adipocytes. Experiments with various RAR agonists and a RAR antagonist in mouse cells demonstrated that the stimulatory effect of ATRA on UCP1 gene expression was indeed mediated by RARs. Consistently, a PPAR? agonist was without effect. Moreover, the ATRA-mediated induction of UCP1 expression in mouse adipocytes was independent of PPAR? coactivator-1?. Conclusions UCP1 expression is differently affected by ATRA in mouse and human adipocytes. ATRA induces UCP1 expression in mouse adipocytes through activation of RARs, whereas expression of UCP1 in human adipocytes is not increased by exposure to ATRA. PMID:24059847
Stereoselectivity for interactions of agonists and antagonists at mouse, rat and human beta3-adrenoceptors.
Popp, Ben D; Hutchinson, Dana S; Evans, Bronwyn A; Summers, Roger J
This study examines the stereoselectivity profile of recombinant mouse, rat and human beta(3)-adrenoceptors expressed in Chinese Hamster Ovary (CHO-K1) cells using radioligand binding, in comparison with endogenously expressed beta(3)-adrenoceptors mediating relaxation responses in mouse ileum. The enantiomeric ratios for several beta-adrenoceptor agonists and antagonists at the cloned mouse, rat and human beta(3)-adrenoceptor were less than those reported at the cloned beta(1)-/beta(2)-adrenoceptor but higher than those reported in previous studies. The degree of stereoselectivity was relatively low for the agonists isoprenaline and noradrenaline but higher for antagonists and, in particular, tertatolol and propranolol. In mouse ileum, stereoselectivity of propranolol and tertatolol was observed under beta(1)-/beta(2)-adrenoceptor blockade. The (-)-enantiomers of propranolol and tertatolol were more effective at antagonism of (-)-isoprenaline-mediated relaxation of mouse ileum than their (+)-enantiomers. The recombinant mouse, rat and human beta(3)-adrenoceptors display stereoselective interactions for agonists and antagonists similar to the stereoselective profile of beta(3)-adrenoceptors in mouse ileum. The degree of stereoselectivity varied between species and the human beta(3)-adrenoceptor displayed higher affinities and enantiomeric ratios than the mouse or rat beta(3)-adrenoceptors. PMID:14744619
The Mouse Genome Database: Genotypes, Phenotypes, and Models of Human Disease
Bult, Carol J.; Eppig, Janan T.; Blake, Judith A.; Kadin, James A.; Richardson, Joel E.
The laboratory mouse is the premier animal model for studying human biology because all life stages can be accessed experimentally, a completely sequenced reference genome is publicly available and there exists a myriad of genomic tools for comparative and experimental research. In the current era of genome scale, data-driven biomedical research, the integration of genetic, genomic and biological data are essential for realizing the full potential of the mouse as an experimental model. The Mouse Genome Database (MGD; http://www.informatics.jax.org), the community model organism database for the laboratory mouse, is designed to facilitate the use of the laboratory mouse as a model system for understanding human biology and disease. To achieve this goal, MGD integrates genetic and genomic data related to the functional and phenotypic characterization of mouse genes and alleles and serves as a comprehensive catalog for mouse models of human disease. Recent enhancements to MGD include the addition of human ortholog details to mouse Gene Detail pages, the inclusion of microRNA knockouts to MGD�s catalog of alleles and phenotypes, the addition of video clips to phenotype images, providing access to genotype and phenotype data associated with quantitative trait loci (QTL) and improvements to the layout and display of Gene Ontology annotations. PMID:23175610
Genetic mapping of the human and mouse phospholipase C genes
M. S. Lyu; D. J. Park; S. G. Rhee; C. A. Kozak
To determine chromosome positions for 10 mouse phospholipase C (PLC) genes, we typed the progeny of two sets of genetic crosses\\u000a for inheritance of restriction enzyme polymorphisms of each PLC. Four mouse chromosomes, Chr 1, 11, 12, and 19, contained\\u000a single PLC genes. Four PLC loci, Plcb1, Plcb2, Plcb4, and Plcg1, mapped to three sites on distal mouse Chr 2.
The mouse and human excitatory amino acid transporter gene (EAAT1) maps to mouse chromosome 15 and a region of syntenic homology on human chromosome 5
Kirschner, M.A.; Arriza, J.L.; Amara, S.G. [Oregon Health Sciences Univ., Portland, OR (United States)] [and others] [Oregon Health Sciences Univ., Portland, OR (United States); and others
The gene for human excitatory amino acid transporter (EAAT1) was localized to the distal region of human chromosome 5p13 by in situ hybridization of metaphase chromosome spreads. Interspecific backcross analysis identified the mouse Eaat1 locus in a region of 5p13 homology on mouse chromosome 15. Markers that are linked with EAAT1 on both human and mouse chromosomes include the receptors for leukemia inhibitory factor, interleukin-7, and prolactin. The Eaat1 locus appears not be linked to the epilepsy mutant stg locus, which is also on chromosome 15. The EAAT1 locus is located in a region of 5p deletions that have been associated with mental retardation and microcephaly. 22 refs., 2 figs.
Epidermal surface antigen (MS17S1) is highly conserved between mouse and human
Cho, Y.J.; Chema, D.; Cho, M. [Univ. of Texas Medical School, Houston, TX (United States)] [and others] [Univ. of Texas Medical School, Houston, TX (United States); and others
A mouse monoclonal antibody ECS-1 raised to human keratinocytes detects a 35-kDa epidermal surface antigen (ESA) and causes keratinocyte dissociation in vitro. ECS-1 stains skin of 16-day mouse embryo and 8- to 9-week human fetus. Mouse Esa cDNA encodes a 379-amino-acid protein that is 99.2% identical to the human, differing at only 3 amino acids. The gene (M17S1) was mapped to mouse chromosome 11, highlighting the conserved linkage synteny existing between human chromosome 17 and mouse chromosome 11. Although the nude locus has been mapped to the same region of chromosome 11, no abnormalities in protein, mRNA, or cDNA or genomic sequences were detected in nude mice. However, both nude and control mice were found to have a second Esa mRNA transcript that conserves amino acid sequence and molecular weight. The mouse and human 5{prime} and 3{prime} untranslated sequences are conserved. Similar RNA folding patterns of the 5{prime} untranslated region are predicted despite a 91-bp insertion in the mouse. These data suggest that both the function and the regulation of ESA protein are of importance and that Esa (M17S1) is not the nude locus gene. 42 refs., 7 figs., 3 tabs.
Abundant alkali-sensitive sites in DNA of human and mouse sperm
Singh, N.P.; Danner, D.B.; McCoy, M.T.; Collins, G.D.; Schneider, E.L. (National Inst. on Aging, Baltimore, MD (United States)); Tice, R.R. (Brookhaven National Lab., NY (United States))
The DNA of human and mouse sperm cells was analyzed by single-cell microgel electrophoresis, by agarose gel electrophoresis, and by alkaline elution-three techniques that can detect single-strand DNA breaks and/or labile sites. Under these conditions a surprisingly large number of single-strand DNA breaks, approximately 10{sup 6} to 10{sup 7} per genome, were detected in human and mouse sperm but not in human lymphocytes or in mouse bone marrow cells. These breaks were also present in chicken erythrocyte DNA, which is also highly condensed. These breaks were not observed under neutral pH conditions nor under denaturing conditions not involving alkali, suggesting that these sites are alkali-sensitive and do not represent preexisting single-strand breaks. The high frequency of such sites in sperm from healthy mouse and human donors suggest that they represent a functional characteristic of condensed chromatin rather than DNA damage.
Comparison of a new pmp22 transgenic mouse line with other mouse models and human patients with CMT1A*
Robertson, AM; Perea, J; McGuigan, A; King, RHM; Muddle, JR; Gabre�ls-Festen, AA; Thomas, PK; Huxley, C
Charcot-Marie-Tooth disease type 1A is a dominantly inherited demyelinating disorder of the peripheral nervous system. It is most frequently caused by overexpression of peripheral myelin protein 22 (PMP22), but is also caused by point mutations in the PMP22 gene. We describe a new transgenic mouse model (My41) carrying the mouse, rather than the human, pmp22 gene. The My41 strain has a severe phenotype consisting of unstable gait and weakness of the hind limbs that becomes obvious during the first 3 weeks of life. My41 mice have a shortened life span and breed poorly. Pathologically, My41 mice have a demyelinating peripheral neuropathy in which 75% of axons do not have a measurable amount of myelin. We compare the peripheral nerve pathology seen in My41 mice, which carry the mouse pmp22 gene, with previously described transgenic mice over-expressing the human PMP22 protein and Trembler-J (TrJ) mice which have a P16L substitution. We also look at the differences between CMT1A duplication patients, patients with the P16L mutation and their appropriate mouse models. PMID:12090404
Conserved mechanisms across development and tumorigenesis revealed by a mouse development perspective of human cancers
Alvin T. Kho; Qing Zhao; Zhaohui Cai; Atul J. Butte; John Y. H. Kim; Scott L. Pomeroy; David H. Rowitch; Isaac S. Kohane
Identification of common mechanisms underlying organ development and primary tumor formation should yield new insights into tumor biology and facilitate the generation of relevant cancer models. We have developed a novel method to project the gene expression profiles of medulloblastomas (MBs)�human cerebellar tumors�onto a mouse cerebellar development sequence: postnatal days 1-60 (P1-P60). Genomically, human medulloblastomas were closest to mouse P1-P10
Peroxisomes in mouse and human lung: their involvement in pulmonary lipid metabolism
Srikanth Karnati; Eveline Baumgart-Vogt
Only sparse information is available from the literature on the peroxisomal compartment and its enzyme composition in mouse\\u000a and human lungs. Therefore, in the present investigation we have characterized peroxisomes in different cell types of adult\\u000a mouse (C57BL\\/6J) and human lungs in a comprehensive study using a variety of light-, fluorescence- and electron microscopic\\u000a as well as biochemical techniques and
Evolutionary changes of the number of olfactory receptor genes in the human and mouse lineages
Nei, Masatoshi
that humans lost many functional OR genes after the human�mouse divergence (HMD) or that mice gained many new OR genes after the HMD whereas ~430 OR genes in the MRCA have become pseudogenes or eliminated.V. All rights reserved. doi:10.1016/j.gene.2004.09.027 Abbreviations: OR, olfactory receptor; HMD, human
LPS and IL-1 differentially activate mouse and human astrocytes: role of CD14
Tarassishin, Leonid; Suh, Hyeon-Sook; Lee, Sunhee C.
Treatment of cultures with toll-like receptor (TLR) ligands or cytokines has become a popular approach to investigate astrocyte neuroinflammatory responses and to simulate the neural environment in various CNS disorders. However, despite much effort, the mechanism of astrocyte activation such as their responses to the TLR ligands and IL-1 remain highly debated. We compared highly pure primary mouse and human astrocyte cultures in their ability to produce proinflammatory mediators (termed �A1�) and immunoregulatory mediators (termed �A2�) in response to LPS, poly IC and IL-1 stimulation. In human astrocytes, IL-1 induced both A1 and A2 responses, poly IC induced mostly A2, and LPS induced neither. In mouse astrocytes, LPS induced mostly an A1 predominant response, poly IC induced both A1 and A2, and IL-1 neither. In addition, mouse astrocytes produce abundant IL-1 protein while human astrocytes did not, despite robust IL-1 mRNA expression. Of the TLR4 receptor complex proteins, human astrocytes expressed TLR4 and MD2 but not CD14, while mouse astrocytes expressed all three. Mouse astrocyte CD14 (cell-associated and soluble) was potently upregulated by LPS. Silencing TLR4 or CD14 by siRNA suppressed LPS responses in mouse astrocytes. In vivo, astrocytes in LPS-injected mouse brains also expressed CD14. Our results show striking differences between human and mouse astrocytes in the use of TLR/IL-1R and subsequent downstream signaling and immune activation. IL-1 translational block in human astrocytes may be a built-in mechanism to prevent autocrine and paracrine cell activation and neuroinflammation. These results have important implications for translational research of human CNS diseases. PMID:24659539
Differences in gene expression between mouse and human for dynamically regulated genes in early embryo.
Madissoon, Elo; T�h�nen, Virpi; Vesterlund, Liselotte; Katayama, Shintaro; Unneberg, Per; Inzunza, Jose; Hovatta, Outi; Kere, Juha
Infertility is a worldwide concern that can be treated with in vitro fertilization (IVF). Improvements in IVF and infertility treatment depend largely on better understanding of the molecular mechanisms for human preimplantation development. Several large-scale studies have been conducted to identify gene expression patterns for the first five days of human development, and many functional studies utilize mouse as a model system. We have identified genes of possible importance for this time period by analyzing human microarray data and available data from online databases. We selected 70 candidate genes for human preimplantation development and investigated their expression in the early mouse development from oocyte to the 8-cell stage. Maternally loaded genes expectedly decreased in expression during development both in human and mouse. We discovered that 25 significantly upregulated genes after fertilization in human included 13 genes whose orthologs in mouse behaved differently and mimicked the expression profile of maternally expressed genes. Our findings highlight many significant differences in gene expression patterns during mouse and human preimplantation development. We also describe four cancer-testis antigen families that are also highly expressed in human embryos: PRAME, SSX, GAGE and MAGEA. PMID:25089626
Cloning the mouse homologue of the human lysosomal acid {alpha}-glucosidase gene
Ding, J.H.; Yang, B.Z.; Liu, H.M. [Duke Univ. Medical Center, Durham, NC (United States)] [and others
Pompe disease (GSD II) is an autosomal recessive disorder caused by a deficiency of lysosomal acid {alpha}-glucosidase (GAA). In an attempt to create a mouse model for Pompe disease, we isolated and characterized the gene encoding the mouse homologue of the human GAA. Twenty clones that extend from exon 2 to the poly(A) tail were isolated from a mouse liver cDNA library, but the remainder of the mRNA proved difficult to obtain by conventional cDNA library screening. Sequences spanning exons 1-2 were cloned by RACE from mouse liver RNA. The full-length liver GAA cDNA contains 3365 nucleotides with a coding region of 2859 nucleotides and a 394 base pair 3{prime}-nontranslated region. The deduced amino acid sequence of the mouse GAA shows 84% identity to the human GAA. Southern blot analysis demonstrated that the mouse GAA was encoded by a single copy gene. Then six bacteriophages containing DNA from the GAA gene were isolated by screening 10{sup 6} phage plaques of a mouse 129 genomic library using a mouse GAA cDNA as a probe. From one of these bacteriophages, an 11-kilobase EcoRI fragment containing exons 3 to 15 was subcloned and sequenced. Work is in progress using this genomic clone to disrupt the GAA gene in murine embryonic stem cells in order to create GSD II mice.
Radial glia require PDGFD-PDGFR? signalling in human but not mouse neocortex.
Lui, Jan H; Nowakowski, Tomasz J; Pollen, Alex A; Javaherian, Ashkan; Kriegstein, Arnold R; Oldham, Michael C
Evolutionary expansion of the human neocortex underlies many of our unique mental abilities. This expansion has been attributed to the increased proliferative potential of radial glia (RG; neural stem cells) and their subventricular dispersion from the periventricular niche during neocortical development. Such adaptations may have evolved through gene expression changes in RG. However, whether or how RG gene expression varies between humans and other species is unknown. Here we show that the transcriptional profiles of human and mouse neocortical RG are broadly conserved during neurogenesis, yet diverge for specific signalling pathways. By analysing differential gene co-expression relationships between the species, we demonstrate that the growth factor PDGFD is specifically expressed by RG in human, but not mouse, corticogenesis. We also show that the expression domain of PDGFR?, the cognate receptor for PDGFD, is evolutionarily divergent, with high expression in the germinal region of dorsal human neocortex but not in the mouse. Pharmacological inhibition of PDGFD-PDGFR? signalling in slice culture prevents normal cell cycle progression of neocortical RG in human, but not mouse. Conversely, injection of recombinant PDGFD or ectopic expression of constitutively active PDGFR? in developing mouse neocortex increases the proportion of RG and their subventricular dispersion. These findings highlight the requirement of PDGFD-PDGFR? signalling for human neocortical development and suggest that local production of growth factors by RG supports the expanded germinal region and progenitor heterogeneity of species with large brains. PMID:25391964
Human embryonic stem cells with biological and epigenetic characteristics similar to those of mouse ESCs
Hanna, Jacob; Cheng, Albert W.; Saha, Krishanu; Kim, Jongpil; Lengner, Christopher J.; Soldner, Frank; Cassady, John P.; Muffat, Julien; Carey, Bryce W.; Jaenisch, Rudolf
Human and mouse embryonic stem cells (ESCs) are derived from blastocyst-stage embryos but have very different biological properties, and molecular analyses suggest that the pluripotent state of human ESCs isolated so far corresponds to that of mouse-derived epiblast stem cells (EpiSCs). Here we rewire the identity of conventional human ESCs into a more immature state that extensively shares defining features with pluripotent mouse ESCs. This was achieved by ectopic induction of Oct4, Klf4, and Klf2 factors combined with LIF and inhibitors of glycogen synthase kinase 3? (GSK3?) and mitogen-activated protein kinase (ERK1/2) pathway. Forskolin, a protein kinase A pathway agonist which can induce Klf4 and Klf2 expression, transiently substitutes for the requirement for ectopic transgene expression. In contrast to conventional human ESCs, these epigenetically converted cells have growth properties, an X-chromosome activation state (XaXa), a gene expression profile, and a signaling pathway dependence that are highly similar to those of mouse ESCs. Finally, the same growth conditions allow the derivation of human induced pluripotent stem (iPS) cells with similar properties as mouse iPS cells. The generation of validated �na�ve� human ESCs will allow the molecular dissection of a previously undefined pluripotent state in humans and may open up new opportunities for patient-specific, disease-relevant research. PMID:20442331
Human ?2-glycoprotein I attenuates mouse intestinal ischemia/reperfusion induced injury and inflammation
Tomasi, Maurizio; Hiromasa, Yasuaki; Pope, Michael R.; Gudlur, Sushanth; Tomich, John M.; Fleming, Sherry D.
Intestinal ischemia-reperfusion (IR)-induced injury results from a complex cascade of inflammatory components. In the mouse model of intestinal IR, the serum protein, ?2-glycoprotein I (?2-GPI) binds to the cell surface early in the cascade. The bound ?2-GPI undergoes a conformational change which exposes a neoantigen recognized by naturally occurring antibodies and initiates the complement cascade. We hypothesized that providing additional antigen with exogenous ?2-GPI would alter IR-induced tissue injury. Administration of human but not mouse ?2-GPI attenuated IR-induced tissue damage and prostaglandin E2 production indicating a physiological difference between ?2-GPI isolated from the two species. To investigate whether structural features were responsible for this physiological difference, we compared the chemical, physical and biochemical properties of the two proteins. Despite possessing 76% amino acid identity and 86% sequence homology, we found that mouse ?2-GPI differs from the human protein in size, carbohydrate chain location, heterogeneity and secondary structural content. These data suggest that the structural differences result in mouse Ab recognition of soluble human but not mouse ?2-GPI and attenuated IR-induced injury. We conclude that caution should be exercised in interpreting results obtained by using human ?2-GPI in a mouse model. PMID:22750067
Human ?2-glycoprotein I attenuates mouse intestinal ischemia/reperfusion induced injury and inflammation.
Tomasi, Maurizio; Hiromasa, Yasuaki; Pope, Michael R; Gudlur, Sushanth; Tomich, John M; Fleming, Sherry D
Intestinal ischemia-reperfusion (IR)-induced injury results from a complex cascade of inflammatory components. In the mouse model of intestinal IR, the serum protein, ?2-glycoprotein I (?2-GPI) binds to the cell surface early in the cascade. The bound ?2-GPI undergoes a conformational change which exposes a neoantigen recognized by naturally occurring antibodies and initiates the complement cascade. We hypothesized that providing additional antigen with exogenous ?2-GPI would alter IR-induced tissue injury. Administration of human but not mouse ?2-GPI attenuated IR-induced tissue damage and prostaglandin E(2) production indicating a physiological difference between ?2-GPI isolated from the two species. To investigate whether structural features were responsible for this physiological difference, we compared the chemical, physical and biochemical properties of the two proteins. Despite possessing 76% amino acid identity and 86% sequence homology, we found that mouse ?2-GPI differs from the human protein in size, carbohydrate chain location, heterogeneity and secondary structural content. These data suggest that the structural differences result in mouse Ab recognition of soluble human but not mouse ?2-GPI and attenuated IR-induced injury. We conclude that caution should be exercised in interpreting results obtained by using human ?2-GPI in a mouse model. PMID:22750067
Species-Specific Metastasis of Human Tumor Cells in the Severe Combined Immunodeficiency Mouse Engrafted with Human Tissue
NASA Astrophysics Data System (ADS)
Shtivelman, Emma; Namikawa, Reiko
We have attempted to model human metastatic disease by implanting human target organs into the immunodeficient C.B-17 scid/scid (severe combined immunodeficiency; SCID) mouse, creating SCID-hu mice. Preferential metastasis to implants of human fetal lung and human fetal bone marrow occurred after i.v. injection of human small cell lung cancer (SCLC) cells into SCID-hu mice; the homologous mouse organs were spared. Clinically more aggressive variant SCLC cells metastasized more efficiently to human fetal lung implants than did cells from classic SCLC. Metastasis of variant SCLC to human fetal bone marrow was enhanced in SCID-hu mice exposed to ?-irradiation or to interleukin 1?. These data indicate that the SCID-hu mice may provide a model in which to study species- and tissue-specific steps of the human metastatic process.
Web-based access to mouse models of human cancers: the Mouse Tumor Biology (MTB) Database
Carol J. Bult; Debra M. Krupke; Dieter N�f; John P. Sundberg; Janan T. Eppig
The Mouse Tumor Biology (MTB) Database serves as a curated, integrated resource for information about tumor genetics and pathology in genetically defined strains of mice (i.e., inbred, transgenic and targeted mutation strains). Sources of information for the database include the published scientific literature and direct data submissions by the scientific community. Researchers access MTB using Web- based query forms and
[Optimization of conditions for human erythropoiesis in human-mouse xenotransplant model].
Zheng, Wei-Wei; Xu, Fei-Fei; Yin, Rong-Hua; Zhan, Yi-Qun; Yang, Xiao-Ming; Li, Zhang-Yan
This study was purposed to investigate the conditions for improving human-mouse xenograft and the erythroid differentiation of human hematopoietic stem cells (HSC) in the xenotransplant model. The engraftments of different mouse strains (NOD/SCID or NOD/SCID/IL2r?(null)), schemes of irradiation (single-time or 2-times radiation; Co(60)?-ray or X-ray) and strategies of CB CD34(+) cells ex vivo culture time and lentivirus infection were compared. The results showed that at 4 weeks after transplantation, the ratio of hCD45 positive cells in bone marrow of NOD/SCID/IL2r?(null) mice increased to (51.4 � 13.9)%, and erythroid precursor could be detected. All of the mice receiving X-ray irradiation for 2 times (a dose of 1 Gy, then the second of 1.5 Gy, with an interval of 15 min) survived. Fresh isolated CB CD34(+) cells were cultured and infected with lentivirus for 72 h and then transplanted into receptor mouse. After 4 weeks, higher engraftment [hCD45 (51.4 � 13.9)%] and better erythroid development [hCD71(+) GPA(+) (5.98 � 3.46)%] were observed. It is concluded that NOD/SCID/IL2r?(null) mice receiving X-ray irradiation for 2 times and were injected with fresh isolated CB CD34(+) cells cultured and infected with lentivirus ex vivo within 72 h show a better xenograft and erythroid development. PMID:24370057
LRP1 Receptor Controls Adipogenesis and Is Up-Regulated In Human and Mouse Obese Adipose Tissue
Paris-Sud XI, Universit� de
LRP1 Receptor Controls Adipogenesis and Is Up- Regulated In Human and Mouse Obese Adipose Tissue human adipose tissues. Interestingly, silencing of LRP1 in fully-differentiated adipocytes also reduces. (2009) LRP1 Receptor Controls Adipogenesis and Is Up-Regulated In Human and Mouse Obese Adipose Tissue
Humanized FcRn mouse models for evaluating pharmacokinetics of human IgG antibodies.
Proetzel, Gabriele; Roopenian, Derry C
A key element for the successful development of novel therapeutic antibodies is to fully understand their pharmacokinetic and pharmacodynamic behavior before performing clinical trials. While many in vitro modeling approaches exist, these simply cannot substitute for data obtained from appropriate animal models. It was established quite early that the unusual long serum half-life of immunoglobulin G's (IgGs) and Fc domains are due to their rescue and recycling by the neonatal Fc receptor (FcRn). The diverse roles of FcRn became apparent after isolation and cloning. Interesting are the significant species differences between rodent and human FcRn reactivity, rendering wild type rodents an inadequate model for studying IgG serum half-life. With the advance of genetic engineering mouse models have been established expressing human FcRn, and lacking mouse FcRn protein. These models have become highly relevant tools for serum half-life analysis of Fc-containing compounds. PMID:23867339
Mouse Models of Human Bladder Cancer as a Tool for Drug Discovery
Seager, Catherine; Puzio-Kuter, Anna M.; Cordon-Cardo, Carlos; McKiernan, James; Abate-Shen, Cory
Muscle-invasive bladder cancer is a deadly condition in dire need of effective new treatments. This unit contains a description of mouse models suitable for the evaluation of potential new therapies. Included is a genetically engineered mouse model of bladder cancer generated by the delivery of an adenovirus expressing Cre recombinase into the bladder lumen. Also described is an orthotopic mouse model created by the instillation of human bladder tumor cells into the bladder lumen of immune deficient mice. Protocols are also provided on the use of these models for the preclinical evaluation of new chemical entities, with mTOR inhibitors shown as an example. PMID:22294368
Comparison of human and mouse T-cell receptor variable gene segment subfamilies
Clark, S.P. [Amgen Center, Thousand Oaks, CA (United States); Arden, B.; Kabelitz, D. [Paul-Ehrlich-Inst., Langen (Germany); Mak, T.W. [Amgen Inst., Toronto, Ontario (Canada)
Like the immunoglobulin Igh-V and Igk-V gene families, the human or mouse TCRV gene families may be grouped into subfamilies displaying {ge} 75% nucleic acid sequence similarity among their members. Systematic interspecies sequence comparisons reveal that most mouse Tcr-V subfamilies exhibit clear homology to human TCRV subfamilies ({ge}60% amino acid sequence similarity). Homologous paris of TCRV genes in mice and humans show higher sequence similarity than TCRV genes from different subfamilies within either species, indicating trans-species evolution of TCRV genes. Mouse and human homologues show conservation of their relative map order, particularly in the 3{prime} region and a similar sequential and developmentally programmed expression. When the V regions from both species were analyzed together, local length differences and conserved residues in the loop regions were revealed, characteristic of each of the four TCRV families. 31 refs., 4 figs.
Enhanced results in mouse and human embryo culture using a modified human tubal fluid medium lacking glucose and phosphate
Patrick Quinn
Purpose: Because various recent studies in both human and laboratory animals have indicated an influential role of glucose and phosphate in embryonic development, this study was initiated to assess the effect of culture medium lacking glucose and phosphate on human IVF. Results: Initial studies with mouse zygotes indicated a significant improvement in embryonic development in both a 171 hybrid and
ERF: Genomic organization, chromosomal localization and promoter analysis of the human and mouse genes
Derong Liu; Elias Pavlopoulos; William Modi; Nickolas Moschonas; George Mavrothalassitis
ERF (Ets2 Repressor Factor) is a ubiquitously expressed ets-domain protein that exhibits strong transcriptional repressor activity, has been shown to suppress ets-induced transformation and has been suggested to be regulated by MAPK phosphorylation. We report here the sequence of the mouse gene, the genomic organization of the human and the mouse genes, their chromosomal position and the analysis of the
Transcript catalogs of human chromosome 21 and orthologous chimpanzee and mouse regions
Xiaolu Sturgeon; Katheleen J. Gardiner
A comprehensive representation of the gene content of the long arm of human chromosome 21 (Hsa21q) remains of interest for\\u000a the study of Down syndrome, its associated phenotypic features, and mouse models. Here we compare transcript catalogs for\\u000a Hsa21q, chimpanzee chromosome 21 (Ptr21q), and orthologous regions of mouse chromosomes 16, 17, and 10 for open reading frame\\u000a (ORF) characteristics and
nature genetics volume 26 october 2000 225 Human-mouse genome comparisons to locate
Zhang, Nancy R.
these results to the human genome: first, it has been unclear how many model organism genomes will be needed a successful expansion of laboratory studies to a genome-scale. Pattern-based computational approaches911 canletter nature genetics · volume 26 · october 2000 225 Human-mouse genome comparisons to locate
Shed Snake Skin and Hairless Mouse Skin as Model Membranes for Human Skin During Permeation Studies
Pauline Carole Rigg; Brian William Barry
Difficulties in obtaining and using human skin have tempted many workers to employ animal membranes for percutaneous absorption studies. We have investigated the suitability of two species of snake (Elaphe obsoleta, Python molurus) for this purpose and compared our in vitro experimental results for human skin and for hairless mouse, a currently popular model. The effects of long-term hydration on
New humanized mouse model of bronchiolitis obliterans syndrome.
Anegon, Ignacio
Humanized animals are transplanted with human tissues and cells to study their behavior as they do in the human body. This commentary briefly summarizes the recent developments and discusses the limitations of these humanized animal models. PMID:25695785
Human and Mouse Mononuclear Phagocyte Networks: A Tale of Two Species?
Reynolds, Gary; Haniffa, Muzlifah
Dendritic cells (DCs), monocytes, and macrophages are a heterogeneous population of mononuclear phagocytes that are involved in antigen processing and presentation to initiate and regulate immune responses to pathogens, vaccines, tumor, and tolerance to self. In addition to their afferent sentinel function, DCs and macrophages are also critical as effectors and coordinators of inflammation and homeostasis in peripheral tissues. Harnessing DCs and macrophages for therapeutic purposes has major implications for infectious disease, vaccination, transplantation, tolerance induction, inflammation, and cancer immunotherapy. There has been a paradigm shift in our understanding of the developmental origin and function of the cellular constituents of the mononuclear phagocyte system. Significant progress has been made in tandem in both human and mouse mononuclear phagocyte biology. This progress has been accelerated by comparative biology analysis between mouse and human, which has proved to be an exceptionally fruitful strategy to harmonize findings across species. Such analyses have provided unexpected insights and facilitated productive reciprocal and iterative processes to inform our understanding of human and mouse mononuclear phagocytes. In this review, we discuss the strategies, power, and utility of comparative biology approaches to integrate recent advances in human and mouse mononuclear phagocyte biology and its potential to drive forward clinical translation of this knowledge. We also present a functional framework on the parallel organization of human and mouse mononuclear phagocyte networks. PMID:26124761
Determination of human transferrin concentrations in mouse models of neisserial infection.
Perera, Yasser; Cobas, Karen; Garrido, Yainelis; Nazabal, Consuelo; Brown, Enma; Pajon, Rolando
Transferrin constitutes the major protein involved in the transport of iron from the sites of absorption to the sites of storage and utilization. Despite the high affinity of transferrin for iron, most bacterial pathogens, such as the human restricted Neisseria meningitidis, have developed iron acquisition mechanisms. Several animal models of bacterial infection that include the exogenous supply of human transferrin have been implemented, and tests using transgenic mouse models are underway. Here we describe an ELISA sandwich procedure based on two monoclonal antibodies with negligible cross-reactivity to murine transferrin, to estimate human transferrin concentrations in mouse sera. The assay can detect as little as 10 ng/ml of human transferrin with coefficients of variation ranging from 1.6% to 4.4% (intra-assay) and 3.8% to 5% (inter-assay). The recovery values range from 90% to 110% in the assay working range (25-400 ng/ml). Human transferrin concentrations estimated in sera from 41 human transferrin transgenic mice ranged from 2 to 14 microg/ml. Further estimations of human transferrin levels in mouse sera of a previously described mouse model of N. meningitidis were also carried out. The intraperitoneal injection of 8 mg of human transferrin achieved a sustained value of human transferrin in mouse sera in the range of 1-2mg/ml over the first 24h, indicating that bacteria reaching the blood stream during this time would be exposed to levels of hTf found in normal human serum. PMID:16529768
Transcript Profiling Identifies Iqgap2?/? Mouse as a Model for Advanced Human Hepatocellular Carcinoma
Gnatenko, Dmitri V.; Xu, Xiao; Zhu, Wei; Schmidt, Valentina A.
It is broadly accepted that genetically engineered animal models do not always recapitulate human pathobiology. Therefore identifying best-fit mouse models of human cancers that truly reflect the corresponding human disease is of vital importance in elucidating molecular mechanisms of tumorigenesis and developing preventive and therapeutic approaches. A new hepatocellular carcinoma (HCC) mouse model lacking a novel putative tumor suppressor IQGAP2 has been generated by our laboratory. The aim of this study was to obtain the molecular signature of Iqgap2?/? HCC tumors and establish the relevance of this model to human disease. Here we report a comprehensive transcriptome analysis of Iqgap2?/? livers and a cross-species comparison of human and Iqgap2?/? HCC tumors using Significance Analysis of Microarray (SAM) and unsupervised hierarchical clustering analysis. We identified the Wnt/?-catenin signaling pathway as the top canonical pathway dysregulated in Iqgap2?/? livers. We also demonstrated that Iqgap2?/? hepatic tumors shared genetic signatures with HCC tumors from patients with advanced disease as evidenced by a 78% mouse-to-human microarray data set concordance rate with 117 out of 151 identified ortholog genes having similar expression profiles across the two species. Collectively, these results indicate that the Iqgap2 knockout mouse model closely recapitulates human HCC at the molecular level and supports its further application for the study of this disease. PMID:23951254
Mapping of the ARIX homeodomain gene to mouse chromosome 7 and human chromosome 11q13
Johnson, K.R. [Jackson Lab., Bar Harbor, ME (United States)] [Jackson Lab., Bar Harbor, ME (United States); Smith, L.; Rhodes, J. [Oregon Health Sciences Univ., Portland, OR (United States)] [and others] [Oregon Health Sciences Univ., Portland, OR (United States); and others
The recently described homeodomain protein ARIX is expressed specifically in noradreneric cell types of the sympathetic nervous system, brain, and adrenal medulla. ARIX interacts with regulatory elements of the genes encoding the noradrenergic biosynthetic enzymes tyrosine hydroxylase and dopamine {beta}-hydroxylase, suggesting a role for ARIX in expression of the noradrenergic phenotype. In the study described here, the mouse and human ARIX genes are mapped. Using segregation analysis of two panels of mouse backcross DNA, mouse Arix was positioned approximately 50 cM distal to the centromere of chromosome 7, near Hbb. Human ARIX was positioned through analysis of somatic cell hybrids and fluorescence in situ hybridization of human metaphase chromosomes to chromosome 7, near Hbb. Human ARIX was positioned through analysis of somatic cell hybrids and fluorescence in situ hybridization of human metaphase chromosomes to chromosome 11q13.3-q13.4. These map locations extend and further define regions of conserved synteny between mouse and human genomes and identify a new candidate gene for inherited developmental disorders linked to human 11q13.
Characterization of Transgenic Mouse Model of Humanized Sialidase
Ghada A. Tarbah
Ganglosidoses are a group of fatal neurological disorders which primarily affect children. Pathologically, these disorders need more characterization. The recent development of mouse models of these diseases makes it possible to identify progressive pathological changes within the CNS at the molecular level. Tay Sachs disease is a lysosomal storage disorder characterized by accumulation of GM2 Ganglioside. Targeted Knockout of ?-hexosaminidase
Antibody engineering via genetic engineering of the mouse: XenoMouse strains are a vehicle for the facile generation of therapeutic human monoclonal antibodies.
Green, L L
The major impediment to the development of murine monoclonal antibodies (mAbs) for therapy in humans has been the difficulty in reducing their potential immunogenicity. XenoMouse�trade mark omitted� mice obviate this problem while retaining the relative ease of generating mAbs from a mouse. XenoMouse strains include germline-configured, megabase-sized YACs carrying portions of the human IgH and Igkappa loci, including the majority of the variable region repertoire, the genes for Cmicro, Cdelta and either Cgamma1, Cgamma2, or Cgamma4, as well as the cis elements required for their function. The IgH and Igkappa transgenes were bred onto a genetic background deficient in production of murine immunoglobulin. The large and complex human variable region repertoire encoded on the Ig transgenes in XenoMouse strains support the development of large peripheral B cell compartments and the generation of a diverse primary immune repertoire similar to that from adult humans. Immunization of XenoMouse mice with human antigens routinely results in a robust secondary immune response, which can ultimately be captured as a large panel of antigen-specific fully human IgGkappa mAbs of sub-nanomolar affinities. Monoclonal antibodies from XenoMouse animals have been shown to have therapeutic potential both in vitro and in vivo, and appear to have the pharmacokinetics of normal human antibodies based on human clinical trials. The utility of XenoMouse strains for the generation of large panels of high-affinity, fully human mAbs can be made available to researchers in the academic and private sectors, and should accelerate the development and application of mAbs as therapeutics for human disease. PMID:10648924
Kit regulates HSC engraftment across the human-mouse species barrier.
Cosgun, Kadriye Nehir; Rahmig, Susann; Mende, Nicole; Reinke, S�ren; Hauber, Ilona; Sch�fer, Carola; Petzold, Anke; Weisbach, Henry; Heidkamp, Gordon; Purbojo, Ariawan; Cesnjevar, Robert; Platz, Alexander; Bornh�user, Martin; Schmitz, Marc; Dudziak, Diana; Hauber, Joachim; Kirberg, J�rg; Waskow, Claudia
In-depth analysis of the cellular and molecular mechanisms regulating human HSC function will require a surrogate host that supports robust maintenance of transplanted human HSCs in vivo, but the currently available options are problematic. Previously we showed that mutations in the Kit receptor enhance engraftment of transplanted HSCs in the mouse. To generate an improved model for human HSC transplantation and analysis, we developed immune-deficient mouse strains containing Kit mutations. We found that mutation of the Kit receptor enables robust, uniform, sustained, and serially transplantable engraftment of human HSCs in adult mice without a requirement for irradiation conditioning. Using this model, we also showed that differential KIT expression identifies two functionally distinct subpopulations of human HSCs. Thus, we have found that the capacity of this Kit mutation to open up stem cell niches across species barriers has significant potential and broad applicability in human HSC research. PMID:25017720
Automated whole-genome multiple alignment of rat, mouse, and human
Brudno, Michael; Poliakov, Alexander; Salamov, Asaf; Cooper, Gregory M.; Sidow, Arend; Rubin, Edward M.; Solovyev, Victor; Batzoglou, Serafim; Dubchak, Inna
We have built a whole genome multiple alignment of the three currently available mammalian genomes using a fully automated pipeline which combines the local/global approach of the Berkeley Genome Pipeline and the LAGAN program. The strategy is based on progressive alignment, and consists of two main steps: (1) alignment of the mouse and rat genomes; and (2) alignment of human to either the mouse-rat alignments from step 1, or the remaining unaligned mouse and rat sequences. The resulting alignments demonstrate high sensitivity, with 87% of all human gene-coding areas aligned in both mouse and rat. The specificity is also high: <7% of the rat contigs are aligned to multiple places in human and 97% of all alignments with human sequence > 100kb agree with a three-way synteny map built independently using predicted exons in the three genomes. At the nucleotide level <1% of the rat nucleotides are mapped to multiple places in the human sequence in the alignment; and 96.5% of human nucleotides within all alignments agree with the synteny map. The alignments are publicly available online, with visualization through the novel Multi-VISTA browser that we also present.
Activation of proto-oncogenes in human and mouse lung tumors
Reynolds, S.H.; Anderson, M.W. (National Institute of Environmental Health Sciences, Research Triangle Park, NC (United States))
Lung cancer is a leading cause of cancer-related deaths in several nations. Epidemiological studies have indicated that 85% of all lung cancer deaths and 30% of all cancer deaths in the US are associated with tobacco smoking. Various chemicals in tobacco smoke are thought to react with DNA and to ultimately yield heritable mutations. In an effort to understand the molecular mechanisms involved in lung tumorigenesis, the authors have analyzed proto-oncogene activation in a series of human lung tumors from smokers and spontaneously occurring and chemically induced lung tumors in mice. Approximately 86% of the human lung tumors and > 90% of the mouse lung tumors were found to contain activated oncogenes. ras Oncogenes activated by point mutations were detected in many of the human lung adenocarcinomas and virtually all of the mouse lung adenomas and adenocarcinomas. The mutation profiles of the activated K-ras genes detected in the chemically induced mouse lung tumors suggest that the observed mutations result from genotoxic effects of the chemicals. Comparison of the K-ras mutations observed in the human lung adenocarcinomas with mutation profiles observed in the mouse lung tumors suggest that bulky hydrophobic DNA adducts may be responsible for the majority of the mutations observed in the activated human K-ras genes. Other data indicate that approximately 20% of human lung tumors contain potentially novel transforming genes that may also be targets for mutagens in cigarette smoke.
Immunodeficient mouse model for human hematopoietic stem cell engraftment and immune system development
Aryee, Ken-Edwin; Shultz, Leonard D.; Brehm, Michael A.
Summary Immunodeficient mice engrafted with human immune systems provide an exciting model to study human immunobiology in an in vivo setting without placing patients at risk. The essential parameter for creation of these �humanized models� is engraftment of human hematopoietic stem cells (HSC) that will allow optimal development of human immune systems. However there are a number of strategies to generate humanized mice and specific protocols can vary significantly among different laboratories. Here we describe a protocol for the co-implantation of human HSC with autologous fetal liver and thymic tissues into immunodeficient mice to create a humanized model with optimal human T cell development. This model, often referred to as the Thy/Liv or BLT (bone marrow, liver, thymus) mouse, develops a functional human immune system, including HLA-restricted human T cells, B cells and innate immune cells. PMID:25062635
Localization of the synapsin II (SYN2) gene to human chromosome 3 and mouse chromosome 6
Lian Li; Lih-Shen Chin; Greengard, P. [Rockefeller Univ., New York, NY (United States)] [and others] [Rockefeller Univ., New York, NY (United States); and others
The synapsins are a family of four synaptic vesicle-associated proteins, synapsins Ia, Ib, IIa, and IIb, that have been implicated in modulation of neurotransmitter release and in synaptogenesis . They are products from alternative splicing of two distinct genes, the synapsin I and synapsin II genes. The synapsin I (SYN1) gene has been mapped to the X chromosome in human and mouse. In this study, we have determined the chromosomal location of the synapsin II (SYN2) gene in both and human and mouse. 10 refs., 1 fig.
Stereoselective inhibition of human, mouse, and horse cholinesterases by bambuterol enantiomers
Anita Bosak; Ivana Gazi?; Vladimir Vinkovi?; Zrinka Kovarik
Bambuterol is a chiral carbamate and a selective inhibitor of butyrylcholinesterase (BChE, EC 184.108.40.206). In order to relate bambuterol selectivity and stereoselectivity of BChE and acetylcholinesterase (AChE, EC 184.108.40.206) of different species, we studied the inhibition of human, mouse, and horse BChE, as well as AChE of human and mouse by (R)- and (S)-bambuterol. AChE and BChE of all studied
Development of Novel Mouse Hybridomas Producing Monoclonal Antibodies Specific to Human and Mouse Nucleolar Protein SURF-6
Polzikov, Mikhail A.; Kordyukova, Maria Yu.; Zavalishina, Larisa E.; Magoulas, Charalambos
SURF-6 is an evolutionarily conserved nucleolar protein that is important for cell viability; however, its function in mammals still remains uncertain. The aim of this study is to generate monoclonal antibodies to human SURF-6 protein suitable for fundamental and biomedical research. The full-size human SURF-6 was expressed as a recombinant GST-fusion protein and used as an antigen to generate monoclonal antibodies, S79 and S148, specific for SURF-6. The monoclonal antibody produced by hybridoma clone S79 specifically recognizes endogenous SURF-6 by Western and immunofluorescence analyses in various cultured human cells, and by immunohistochemistry in paraffin-embedded sections of human breast cancer samples. Moreover, S79 immunoprecipitates protein complexes containing SURF-6 from HeLa cells extracts. The antibody S79 recognizes SURF-6 only in human cells; however, the antibody produced by hybridoma clone S148 can detect SURF-6 of human and mouse origin. Monoclonal antibodies to the nucleolar protein SURF-6 described in this work can be a useful tool for studies of ribosome biogenesis in normal and cancer cells. PMID:22316485
Development of novel mouse hybridomas producing monoclonal antibodies specific to human and mouse nucleolar protein SURF-6.
Polzikov, Mikhail A; Kordyukova, Maria Yu; Zavalishina, Larisa E; Magoulas, Charalambos; Zatsepina, Olga V
SURF-6 is an evolutionarily conserved nucleolar protein that is important for cell viability; however, its function in mammals still remains uncertain. The aim of this study is to generate monoclonal antibodies to human SURF-6 protein suitable for fundamental and biomedical research. The full-size human SURF-6 was expressed as a recombinant GST-fusion protein and used as an antigen to generate monoclonal antibodies, S79 and S148, specific for SURF-6. The monoclonal antibody produced by hybridoma clone S79 specifically recognizes endogenous SURF-6 by Western and immunofluorescence analyses in various cultured human cells, and by immunohistochemistry in paraffin-embedded sections of human breast cancer samples. Moreover, S79 immunoprecipitates protein complexes containing SURF-6 from HeLa cells extracts. The antibody S79 recognizes SURF-6 only in human cells; however, the antibody produced by hybridoma clone S148 can detect SURF-6 of human and mouse origin. Monoclonal antibodies to the nucleolar protein SURF-6 described in this work can be a useful tool for studies of ribosome biogenesis in normal and cancer cells. PMID:22316485
Color Tuning in Short Wavelength-Sensitive Human and Mouse Visual Pigments: Ab initio Quantum Mechanics/Molecular Mechanics Studies
Yokoyama, Shozo
Color Tuning in Short Wavelength-Sensitive Human and Mouse Visual Pigments: Ab initio Quantum-retinal in human blue and mouse UV cone visual pigments as well as in bovine rhodopsin by hybrid quantum mechanical energies show that UV-sensitive pigments have deprotonated SB nitrogen, while violet-sensitive pigments
A short-term assay utilizing a human/mouse monochromosomal hybrid cell line R3-5, to detect chemically induced aneuploidy in mammalian cells is described. A single human chromosome transferred into mouse cells was used as a cytogenetic marker to quantitate abnormal chromosome seg...
Gene order is conserved within the human chromosome 21 linkage group on mouse chromosome 10
Irving, N.G.; Cabin, D.E.; Swanson, D.A.; Reeves, R.H. (Johns Hopkins Univ. School of Medicine, Baltimore, MD (United States))
One hundred progeny from each of two intersubspecific mouse backcrosses were used to construct a comparative genetic map of a region of mouse chromosome 10 (MMU10) that is homologous to the distal tip of the long arm of human chromosome 21 (HSA21). The analysis included five genes and three simple sequence repeat markers, two of which flanked the HSA21-homologous cluster on either side. Analysis of 200 backcross progeny detected at least one crossover between each pair of adjacent genes and demonstrated that the proximal to distal orientation of the cluster was reversed between human and mouse. The order was determined to be Fyn-1-D10Mit20-S100b-Col6a1-Itgb2-Pfk1/D10Mit7-D10Mit11. Comparative mapping supports the order of corresponding markers on HSA21 determined using pulsed-field gel electrophoresis and radiation hybrid line data. However, sequence tagged site content mapping of human yeast artificial chromosomes (YACs) yielded conflicting data on the relative positions of human COL6A1 and S100B on HSA21. This discrepancy was resolved here by demonstrating that several key YACs used in the human contig analysis were mistyped for S100B. The murine map reported here provides a scaffold for construction of physical maps and yeast artificial chromosome contigs that will be useful in the development of mouse models for the study of Down syndrome. 28 refs., 4 figs., 2 tabs.
Regional assignments of the zinc finger Y-linked gene (ZFY) and related sequences on human and mouse chromosomes.
Leung, W Y; Lindgren, V; Lau, Y F; Yang-Feng, Y L
Recent chromosome walking experiments have identified a candidate gene (ZFY) for the testis-determining factor on the human Y chromosome (Page et al., 1987). We report here the regional assignments of the ZFY gene and related sequences in the human and the mouse. By in situ hybridization, we assigned ZFX and ZFY to human chromosome bands Xp21 and Yp11.3, respectively. Although the mouse harbors two Zfy genes, only one site at band A1 of its Y chromosome was significantly labeled. The mouse Zfx gene and the Zfa gene on chromosome 10 were assigned to bands XD and 10B5, respectively. These assignments of the ZFX gene in human and mouse add another marker to the conserved syntenic group for evaluating the evolutionary relationship of the human and mouse X chromosomes. PMID:2265557
A mouse model of Down syndrome trisomic for all human chromosome 21 syntenic regions
Yu, Tao; Li, Zhongyou; Jia, Zhengping; Clapcote, Steven J.; Liu, Chunhong; Li, Shaomin; Asrar, Suhail; Pao, Annie; Chen, Rongqing; Fan, Ni; Carattini-Rivera, Sandra; Bechard, Allison R.; Spring, Shoshana; Henkelman, R. Mark; Stoica, George; Matsui, Sei-Ichi; Nowak, Norma J.; Roder, John C.; Chen, Chu; Bradley, Allan; Yu, Y. Eugene
Down syndrome (DS) is caused by the presence of an extra copy of human chromosome 21 (Hsa21) and is the most common genetic cause for developmental cognitive disability. The regions on Hsa21 are syntenically conserved with three regions located on mouse chromosome 10 (Mmu10), Mmu16 and Mmu17. In this report, we describe a new mouse model for DS that carries duplications spanning the entire Hsa21 syntenic regions on all three mouse chromosomes. This mouse mutant exhibits DS-related neurological defects, including impaired cognitive behaviors, reduced hippocampal long-term potentiation and hydrocephalus. These results suggest that when all the mouse orthologs of the Hsa21 genes are triplicated, an abnormal cognitively relevant phenotype is the final outcome of the elevated expressions of these orthologs as well as all the possible functional interactions among themselves and/or with other mouse genes. Because of its desirable genotype and phenotype, this mutant may have the potential to serve as one of the reference models for further understanding the developmental cognitive disability associated with DS and may also be used for developing novel therapeutic interventions for this clinical manifestation of the disorder. PMID:20442137
Characterization of photolyase/blue-light receptor homologs in mouse and human cells.
Kobayashi, K; Kanno, S; Smit, B; van der Horst, G T; Takao, M; Yasui, A
We isolated and characterized mouse photolyase-like genes, mCRY1 (mPHLL1) and mCRY2 (mPHLL2), which belong to the photolyase family including plant blue-light receptors. The mCRY1 and mCRY2 genes are located on chromosome 10C and 2E, respectively, and are expressed in all mouse organs examined. We raised antibodies specific against each gene product using its C-terminal sequence, which differs completely between the genes. Immunofluorescent staining of cultured mouse cells revealed that mCRY1 is localized in mitochondria whereas mCRY2 was found mainly in the nucleus. The subcellular distribution of CRY proteins was confirmed by immunoblot analysis of fractionated mouse liver cell extracts. Using green fluorescent protein fused peptides we showed that the C-terminal region of the mouse CRY2 protein contains a unique nuclear localization signal, which is absent in the CRY1 protein. The N-terminal region of CRY1 was shown to contain the mitochondrial transport signal. Recombinant as well as native CRY1 proteins from mouse and human cells showed a tight binding activity to DNA Sepharose, while CRY2 protein did not bind to DNA Sepharose at all under the same condition as CRY1. The different cellular localization and DNA binding properties of the mammalian photolyase homologs suggest that despite the similarity in the sequence the two proteins have distinct function(s). PMID:9801304
MicroRNAs and induced pluripotent stem cells for human disease mouse modeling.
Underbayev, Chingiz; Kasar, Siddha; Yuan, Yao; Raveche, Elizabeth
Human disease animal models are absolutely invaluable tools for our understanding of mechanisms involved in both physiological and pathological processes. By studying various genetic abnormalities in these organisms we can get a better insight into potential candidate genes responsible for human disease development. To this point a mouse represents one of the most used and convenient species for human disease modeling. Hundreds if not thousands of inbred, congenic, and transgenic mouse models have been created and are now extensively utilized in the research labs worldwide. Importantly, pluripotent stem cells play a significant role in developing new genetically engineered mice with the desired human disease-like phenotype. Induced pluripotent stem (iPS) cells which represent reprogramming of somatic cells into pluripotent stem cells represent a significant advancement in research armament. The novel application of microRNA manipulation both in the generation of iPS cells and subsequent lineage-directed differentiation is discussed. Potential applications of induced pluripotent stem cell--a relatively new type of pluripotent stem cells--for human disease modeling by employing human iPS cells derived from normal and diseased somatic cells and iPS cells derived from mouse models of human disease may lead to uncovering of disease mechanisms and novel therapies. PMID:22619497
Prediction of the odorant binding site of olfactory receptor proteins by human-mouse comparisons
Olfactory receptors (ORs) are a large family of proteins involved in the recognition and discrimination of numerous odorants. These receptors belong to the G-protein coupled receptor (GPCR) hyperfamily, for which little structural data are available. In this study we predict the binding site residues of OR proteins by analyzing a set of 1441 OR protein sequences from mouse and human.
Binding of Mouse and Human Fibulin-2 to Extracellular Matrix Ligands
Takako Sasaki; Walter G�hring; Te-Cheng Pan; Mon-Li Chu; Rupert Timpl
Recombinant mouse and human fibulin-2 were obtained as disulfide-bonded trimers from transfected kidney cell clones and used in solid phase, biosensor and radioligand binding assays. Strong binding occurred with fibronectin and required calcium. A distinct interaction was also observed with nidogen but this was only partially blocked by EDTA. Distinctly weaker affinities were detected for collagen IV, perlecan and the
Mouse Models for Deafness: Lessons for the Human Inner Ear and Hearing Loss
Avraham, Karen
Mouse Models for Deafness: Lessons for the Human Inner Ear and Hearing Loss Karen B. Avraham high-resolution com- puted tomography (CT) scanning or invasive sur- gery, most studies on the ear interventions can be developed that can treat the diseased inner ear before permanent damage has occurred
Patterning Mouse and Human Embryonic Stem Cells Using Micro-contact Printing
Zandstra, Peter W.
Chapter 2 Patterning Mouse and Human Embryonic Stem Cells Using Micro-contact Printing Raheem�cell contact. The protocol described here uses micro-contact printing to pattern ECM onto tissue culture and immunohistochemistry. Key words: Micro-contact printing, soft lithography, embryonic stem cell, flow cytometry
Human and mouse gene structure: comparative analysis and application to exon prediction
Serafim Batzoglou; Lior Pachter; Jill P. Mesirov; Bonnie Berger; Eric S. Lander
We describe a novel analytical approach to gene recognition based on cross-species comparison We first undertook a comparison of orthologous genomic look from human and mouse, studying the extent of similarity in the number, size and sequence of exons and introns We then developed an approach for recognizing genes within such orthologous regions, by first aligning the regions using an
Comparison of human and mouse sperm chromatin structure by flow cytometry
Donald P. Evenson; Zbigniew Darzynkiewicz; Myron R. Melamed
Human and mouse sperm nuclei obtained by sonication or mechanical agitation of freshly isolated sperm in the presence of anionic detergent were purified through a sucrose gradient and stained with acridine orange (AO); their fluorescence intensity was measured by flow cytometry. The green fluorescence, characteristic of AO binding to DNA by intercalation, was twice lower per unit of DNA for
Generation of retinal cells from mouse and human induced pluripotent stem cells
Yasuhiko Hirami; Fumitaka Osakada; Kazutoshi Takahashi; Keisuke Okita; Shinya Yamanaka; Hanako Ikeda; Nagahisa Yoshimura; Masayo Takahashi
We previously reported a technique for generating retinal pigment epithelia (RPE) and putative photoreceptors from embryonic stem (ES) cells. Here we tested whether our procedure can promote retinal differentiation of mouse and human induced pluripotent stem cells (iPSCs). Treating iPSCs with Wnt and Nodal antagonists in suspension culture induced expression of markers of retinal progenitor cells and generated RPE cells.
ANTI-Mac-1 SELECTIVELY INHIBITS THE MOUSE AND HUMAN TYPE THREE COMPLEMENT RECEPTOR*
Springer, Timothy A.
ANTI-Mac-1 SELECTIVELY INHIBITS THE MOUSE AND HUMAN TYPE THREE COMPLEMENT RECEPTOR* By DAVID I). The type one complement receptor (CR1) displays specificity for C3b and C4b (14-17). This receptor has been isolated and consists of a single polypeptide chain of 205,000 tool wt (14, 15). The type three complement
Analysis of the heavy metal-responsive transcription factor MTF-1 from human and mouse
Mueller, H.P.; Brugnera, E.; Georgiev, O. [Universitaet Zuerich (Switzerland)] [and others
Heavy metal-induced transcription in mammalian cells is conferred by the metalresponsive 70 kDa transcription factor MTF-1 which contains six zinc fingers and at least three activation domains. In previous cell transfection experiments we have shown that the zinc finger region confers an about 3 fold metal inducibility of transcription, due to its differential zinc binding. However, we also noted that human MTF-1 was more metal-responsive than the mouse factor (about 10 fold versus 3 fold, respectively). Here we analyze this difference in more detail by using chimeric human-mouse factors and narrow the critical region to a 64 amino acid stretch immediately downstream of the zinc fingers, overlapping with the acidic activation domain. A short human segment of this region (aa 313-377) confers efficient metal induction to the mouse MTF-1 when replacing the corresponding mouse region. However, high metal inducibility requires an unaltered MTF-I and is lost when human MTF-I is fused to the general activation domain of herpesvirus VP16 Wild type and truncation mutants of MTF-1 fused to VPI6 yield chimeras of high transcriptional activity, some exceeding the wildtype regulator, but only limited (about 3 fold) heavy metal inducibility. 22 refs., 4 figs.
A Quest for Human and Mouse Embryonic Stem Cell-specific Proteins
Dennis Van Hoof; Robert Passier; Dorien Ward-Van Oostwaard; Martijn W. H. Pinkse; Albert J. R. Heck; Christine L. Mummery; Jeroen Krijgsveld
Embryonic stem cells (ESCs) are of immense interest as they can proliferate indefinitely in vitro and give rise to any adult cell type, serving as a potentially unlimited source for tissue replacement in regenerative medicine. Extensive analyses of numerous human and mouse ESC lines have shown generic similarities and differences at both the tran- scriptional and functional level. However, comprehensive
Expression Profiling of Nuclear Receptors in Human and Mouse Embryonic Stem Cells
Xie, Chang-Qing; Jeong, Yangsik; Fu, Mingui; Bookout, Angie L.; Garcia-Barrio, Minerva T.; Sun, Tingwan; Kim, Bong-hyun; Xie, Yang; Root, Sierra; Zhang, Jifeng; Xu, Ren-He; Chen, Y. Eugene; Mangelsdorf, David J.
Nuclear receptors (NRs) regulate gene expression in essential biological processes including differentiation and development. Here we report the systematic profiling of NRs in human and mouse embryonic stem cell (ESC) lines and during their early differentiation into embryoid bodies. Expression of the 48 human and mouse NRs was assessed by quantitative real-time PCR. In general, expression of NRs between the two human cell lines was highly concordant, whereas in contrast, expression of NRs between human and mouse ESCs differed significantly. In particular, a number of NRs that have been implicated previously as crucial regulators of mouse ESC biology, including ERR?, DAX-1, and LRH-1, exhibited diametric patterns of expression, suggesting they may have distinct species-specific functions. Taken together, these results highlight the complexity of the transcriptional hierarchy that exists between species and governs early development. These data should provide a unique resource for further exploration of the species-specific roles of NRs in ESC self-renewal and differentiation. PMID:19196830
A Lentiviral RNAi Library for Human and Mouse Genes Applied to an
Sabatini, David M.
Resource A Lentiviral RNAi Library for Human and Mouse Genes Applied to an Arrayed Viral High-verified constructs. To test the utility of the library for ar- rayed screens, we developed a screen based on high such as Caenorhabditis elegans and Drosophila melanogaster, the recognition that RNA in- terference (RNAi) can
Bioinformatic Analysis of Retroelement- Associated Sequences in Human and Mouse Promoters
Nadezhda M. Usmanova; Nikolai V. Tomilin
Mammalian genomes contain large number of retroelements (SINEs, LINEs and LTRs) which could affect expression of protein coding genes through associated transcription factor binding sites (TFBS). Activity of the retroelement-associated TFBS in many genes is confirmed experimentally but their global functional impact remains unclear. Human SINEs (Alu repeats) and mouse SINEs (B1 and B2 repeats) are known to be clustered
Mapping GRB2, a signal transduction gene in the human and the mouse
Yulug, I.G.; Egan, S.E.; See, C.G. [Imperial College, London (United Kingdom)] [and others] [Imperial College, London (United Kingdom); and others
The authors have mapped GRB2, a signal transduction gene whose protein product is an essential component of the pathway between tyrosine kinases (such as the epidermal growth factor receptor) and downstream proteins (such as Ras and Sos). They assigned GRB2 to human chromosome 17 by hybridization to a somatic cell hybrid mapping panel. To position the locus at a much finer resolution, they have isolated the human GRB2 gene in three different cosmids, which they have mapped by fluorescence in situ hybridization to the long arm of human chromosome 17 (17q24-q25). They have hybridized a human GRB2 open reading frame probe to mouse DNAs from the European Interspecific Backcross. The segregation patterns reveal that the mouse Grb2 locus maps distally on chromosome 11, and an additional Grb2-related locus is present on chromosome 4 of one of the parental strains, Mus spretus/CRC. 32 refs., 5 figs., 2 tabs.
The effects of oncogene transfection on growth and antibody production of human-mouse heterohybridomas
Otmar Hohenwarter; Andrea Waltenberger; Christine Schmatz; Hermann Katinger
Human-mouse heterohybridomas producing human monoclonal antibodies show slower growth rates and lower peak cell densities than murine hybridomas. In order to improve the growth properties we transfected a heterohybridoma cell line with expression plasmids containing the oncogenes v-src, c-Ha-ras and SV40largeT. The plasmids were transferred by electroporation. Growth promoting activities of the plasmids were proven in NIH3T3 cells whereby a
Reperfusion Injury Intensifies the Adaptive Human T Cell Alloresponse in a Human-Mouse Chimeric Artery Model
Yi, Tai; Fogal, Birgit; Hao, Zhengrong; Tobiasova, Zuzana; Wang, Chen; Rao, Deepak A.; Al-Lamki, Rafia S.; Kirkiles-Smith, Nancy C.; Kulkarni, Sanjay; Bradley, John R.; Bothwell, Alfred L.M.; Sessa, William C.; Tellides, George; Pober, Jordan S.
Objective Peri-operative non-immune injuries to an allograft can decrease graft survival. We have developed a model for studying this process using human materials. Methods and Results Human artery segments were transplanted as infrarenal aortic interposition grafts into an immunodeficient mouse host, allowed to �heal in� for 30 days, and then re-transplanted into a second mouse host. To induce a reperfusion injury, the healed in artery segments were incubated for 3 h under hypoxic conditions ex vivo prior to re-transplant. To induce immunological rejection, the animals receiving the re-transplanted artery segment were adoptively transferred with human peripheral blood mononuclear cells or purified T cells from a donor allogeneic to the artery one week prior to surgery. To compare rejection of injured vs. healthy tissues, these manipulations were combined. Results were analyzed ex vivo by histology, morphometry, immunohistochemistry and mRNA quantitation or in vivo by ultrasound. Our results showed that reperfusion injury, which otherwise heals with minimal sequelae, intensifies the degree of allogeneic T cell-mediated injury to human artery segments. Conclusion We developed a new human-mouse chimeric model demonstrating interactions of reperfusion injury and alloimmunity using human cells and tissues that may be adapted to study other forms of non-immune injury and other types of adaptive immune responses. PMID:22053072
A transgenic mouse model expressing exclusively human hemoglobin E: indications of a mild oxidative stress.
Chen, Qiuying; Fabry, Mary E; Rybicki, Anne C; Suzuka, Sandra M; Balazs, Tatiana C; Etzion, Zipora; de Jong, Kitty; Akoto, Edna K; Canterino, Joseph E; Kaul, Dhananjay K; Kuypers, Frans A; Lefer, David; Bouhassira, Eric E; Hirsch, Rhoda Elison
Hemoglobin (Hb) E (?26 Glu?Lys) is the most common abnormal hemoglobin (Hb) variant in the world. Homozygotes for HbE are mildly thalassemic as a result of the alternate splice mutation and present with a benign clinical picture (microcytic and mildly anemic) with rare clinical symptoms. Given that the human red blood cell (RBC) contains both HbE and excess ?-chains along with minor hemoglobins, the consequence of HbE alone on RBC pathophysiology has not been elucidated. This becomes critical for the highly morbid ?(E)-thalassemia disease. We have generated transgenic mice exclusively expressing human HbE (HbEKO) that exhibit the known aberrant splicing of ?(E) globin mRNA, but are essentially non-thalassemic as demonstrated by RBC ?/? (human) globin chain synthesis. These mice exhibit hematological characteristics similar to presentations in human EE individuals: microcytic RBC with low MCV and MCH but normal MCHC; target RBC; mild anemia with low Hb, HCT and mildly elevated reticulocyte levels and decreased osmotic fragility, indicating altered RBC surface area to volume ratio. These alterations are correlated with a mild RBC oxidative stress indicated by enhanced membrane lipid peroxidation, elevated zinc protoporphyrin levels, and by small but significant changes in cardiac function. The C57 (background) mouse and full KO mouse models expressing HbE with the presence of HbS or HbA are used as controls. In select cases, the HbA full KO mouse model is compared but found to be limited due to its RBC thalassemic characteristics. Since the HbEKO mouse RBC lacks an abundance of excess ?-chains that would approximate a mouse thalassemia (or a human thalassemia), the results indicate that the observed in vivo RBC mild oxidative stress arises, at least in part, from the molecular consequences of the HbE mutation. PMID:22260787
Cocktails of human anti-cancer antibodies show a synergistic effect in nude mouse tumor xenografts.
Glassy, Mark C; McKnight, Michael E; Kotlan, Beatrix; Glassy, Eric F; Koda, Keiji
A panel of four natural human monoclonal IgG antibodies derived from B lymphocytes isolated from regional draining lymph nodes of cancer patients has been developed and characterized. The four human antibodies are termed, RM1, RM2, RM3, and RM4. The immunoreactivity of this panel of four human antibodies is restricted to tumor cells. Individually, these human MAbs show tumor targeting and are effective in inhibiting tumor growth in nude mouse xenograft models. When used in combination the antibodies show an additive effect in slowing down the progression of tumors in xenograft models suggesting that cocktails of antibodies may be useful in the clinic. PMID:18334744
Lambda-Chain Production in Human Lymphoblast-Mouse Fibroblast Hybrids
Orkin, Stuart H.; Buchanan, Philip D.; Yount, William J.; Reisner, Howard; Littlefield, John W.
Mutant human lymphoblast cells deficient in hypoxanthine phosphoribosyltransferase (EC 22.214.171.124) activity were hybridized with thymidine kinase (EC 18.104.22.168)-deficient mouse fibroblasts. Hybrid cells were readily selected, as both parental lines were nonreverting and eliminated by hypoxanthine-amethopterinthymidine medium. Human lambda (?) chain was the only immunoglobulin chain produced by the lymphoblast parent, as determined by immunofluorescent techniques. Two independent hybrid clones chosen for detailed study synthesized human ? chain, and continued to do so after prolonged culture. As in both parental lines, no human immunoglobulin heavy chains, complements C3 or C4, or ?1-antitrypsin, or mouse immunoglobulin chains or complement C5 were detectable in the hybrids. Selection against thymidine kinase-containing hybrid cells with 5-bromodeoxyuridine did not eliminate positive ?-chain reactivity, suggesting that the kinase and ?-chain loci are not linked. The continued production of an immunoglobulin chain by human lymphoblast-mouse fibroblast hybrids contrasts with the extinction of other differentiated functions in several hybrid systems, and indicates that gene localization and linkage analysis for human immunoglobulin chains should be feasible with this system. Images PMID:4599625
Increased amyloidogenic processing of transgenic human APP in X11-like deficient mouse brain
Background X11-family proteins, including X11, X11-like (X11L) and X11-like 2 (X11L2), bind to the cytoplasmic domain of amyloid ?-protein precursor (APP) and regulate APP metabolism. Both X11 and X11L are expressed specifically in brain, while X11L2 is expressed ubiquitously. X11L is predominantly expressed in excitatory neurons, in contrast to X11, which is strongly expressed in inhibitory neurons. In vivo gene-knockout studies targeting X11, X11L, or both, and studies of X11 or X11L transgenic mice have reported that X11-family proteins suppress the amyloidogenic processing of endogenous mouse APP and ectopic human APP with one exception: knockout of X11, X11L or X11L2 has been found to suppress amyloidogenic metabolism in transgenic mice overexpressing the human Swedish mutant APP (APPswe) and the mutant human PS1, which lacks exon 9 (PS1dE9). Therefore, the data on X11-family protein function in transgenic human APP metabolism in vivo are inconsistent. Results To confirm the interaction of X11L with human APP ectopically expressed in mouse brain, we examined the amyloidogenic metabolism of human APP in two lines of human APP transgenic mice generated to also lack X11L. In agreement with previous reports from our lab and others, we found that the amyloidogenic metabolism of human APP increased in the absence of X11L. Conclusion X11L appears to aid in the suppression of amyloidogenic processing of human APP in brain in vivo, as has been demonstrated by previous studies using several human APP transgenic lines with various genetic backgrounds. X11L appears to regulate human APP in a manner similar to that seen in endogenous mouse APP metabolism. PMID:20843325
�H MRS characterization of neurochemical profiles in orthotopic mouse models of human brain tumors.
Hulsey, Keith M; Mashimo, Tomoyuki; Banerjee, Abhishek; Soesbe, Todd C; Spence, Jeffrey S; Vemireddy, Vamsidhara; Maher, Elizabeth A; Bachoo, Robert M; Choi, Changho
Glioblastoma (GBM), the most common primary brain tumor, is resistant to currently available treatments. The development of mouse models of human GBM has provided a tool for studying mechanisms involved in tumor initiation and growth as well as a platform for preclinical investigation of new drugs. In this study we used (1) H MR spectroscopy to study the neurochemical profile of a human orthotopic tumor (HOT) mouse model of human GBM. The goal of this study was to evaluate differences in metabolite concentrations in the GBM HOT mice when compared with normal mouse brain in order to determine if MRS could reliably differentiate tumor from normal brain. A TE =19?ms PRESS sequence at 9.4?T was used for measuring metabolite levels in 12 GBM mice and 8 healthy mice. Levels for 12 metabolites and for lipids/macromolecules at 0.9?ppm and at 1.3?ppm were reliably detected in all mouse spectra. The tumors had significantly lower concentrations of total creatine, GABA, glutamate, total N-acetylaspartate, aspartate, lipids/macromolecules at 0.9?ppm, and lipids/macromolecules at 1.3?ppm than did the brains of normal mice. The concentrations of glycine and lactate, however, were significantly higher in tumors than in normal brain. PMID:25394324
Human NAIP and mouse NAIP1 recognize bacterial type III secretion needle protein for inflammasome activation.
Yang, Jieling; Zhao, Yue; Shi, Jianjin; Shao, Feng
Inflammasome mediated by central nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) protein is critical for defense against bacterial infection. Here we show that type III secretion system (T3SS) needle proteins from several bacterial pathogens, including Salmonella typhimurium, enterohemorrhagic Escherichia coli, Shigella flexneri, and Burkholderia spp., can induce robust inflammasome activation in both human monocyte-derived and mouse bone marrow macrophages. Needle protein activation of human NRL family CARD domain containing 4 (NLRC4) inflammasome requires the sole human neuronal apoptosis inhibitory protein (hNAIP). Among the seven mouse NAIPs, NAIP1 functions as the mouse counterpart of hNAIP. We found that NAIP1 recognition of T3SS needle proteins was more robust in mouse dendritic cells than in bone marrow macrophages. Needle proteins, as well as flagellin and rod proteins from five different bacteria, exhibited differential and cell type-dependent inflammasome-stimulating activity. Comprehensive profiling of the three types of NAIP ligands revealed that NAIP1 sensing of the needle protein dominated S. flexneri-induced inflammasome activation, particularly in dendritic cells. hNAIP/NAIP1 and NAIP2/5 formed a large oligomeric complex with NLRC4 in the presence of corresponding bacterial ligands, and could support reconstitution of the NLRC4 inflammasome in a ligand-specific manner. PMID:23940371
Comparative mapping of human Chromosome 14q11.2-q13 genes with mouse homologous gene regions
Deepak Kamnasaran; Patricia C. M. O'Brien; Malcolm A. Ferguson-Smith; Diane W. Cox
.�� An examination of the synteny blocks between mouse and human chromosomes aids in understanding the evolution of chromosome\\u000a divergence between these two species. We comparatively mapped the human (HSA) Chromosome (Chr) 14q11.2-q13 cytogenetic region\\u000a with the intervals of orthologous genes on mouse (MMU) chromosomes. A lack of conserved gene order was identified between\\u000a the human cytogenetic region and the
Sequence divergence and chromosomal rearrangements during the evolution of human pseudoautosomal genes and their mouse homologs
Ellison, J.; Li, X.; Francke, U. [USCS, San Francisco, CA (United States)] [and others
The pseudoautosomal region (PAR) is an area of sequence identity between the X and Y chromosomes and is important for mediating X-Y pairing during male meiosis. Of the seven genes assigned to the human PAR, none of the mouse homologs have been isolated by a cross-hybridization strategy. Two of these homologs, Csfgmra and II3ra, have been isolated using a functional assay for the gene products. These genes are quite different in sequence from their human homologs, showing only 60-70% sequence similarity. The Csfgmra gene has been found to further differ from its human homolog in being isolated not on the sex chromosomes, but on a mouse autosome (chromosome 19). Using a mouse-hamster somatic cell hybrid mapping panel, we have mapped the II3ra gene to yet another mouse autosome, chromosome 14. Attempts to clone the mouse homolog of the ANT3 locus resulted in the isolation of two related genes, Ant1 and Ant2, but failed to yield the Ant3 gene. Southern blot analysis of the ANT/Ant genes showed the Ant1 and Ant2 sequences to be well-conserved among all of a dozen mammals tested. In contrast, the ANT3 gene only showed hybridization to non-rodent mammals, suggesting it is either greatly divergent or has been deleted in the rodent lineage. Similar experiments with other human pseudoautosomal probes likewise showed a lack of hybridization to rodent sequences. The results show a definite trend of extensive divergence of pseudoautosomal sequences in addition to chromosomal rearrangements involving X;autosome translocations and perhaps gene deletions. Such observations have interesting implications regarding the evolution of this important region of the sex chromosomes.
Inflammation precedes the development of human malignant mesotheliomas in a SCID mouse xenograft model
Hillegass, Jedd M.; Shukla, Arti; Lathrop, Sherrill A.; MacPherson, Maximilian B.; Beuschel, Stacie L.; Butnor, Kelly J.; Testa, Joseph R.; Pass, Harvey I.; Carbone, Michele; Steele, Chad; Mossman, Brooke T.
Asbestos fibers cause chronic inflammation that may be critical to the development of malignant mesothelioma (MM). Two human MM cell lines (Hmeso, PPM Mill) were used in a SCID mouse xenograft model to assess time-dependent patterns of inflammation and tumor formation. After intraperitoneal (IP) injection of MM cells, mice were euthanized at 7, 14, and 30 days, and peritoneal lavage fluid (PLF) was examined for immune cell profiles and human and mouse cytokines. Increases in human MM-derived IL-6, IL-8, bFGF, and VEGF were observed in mice at 7 days postinjection of either MM line, and a striking neutrophilia was observed at all time points. Free-floating tumor spheroids developed in mice at 14 days, and both spheroids and adherent MM tumor masses occurred in all mice at 30 days. Results suggest that inflammation and cytokine production precede and may be critical to the development of MMs. PMID:20716277
Mybl2 (Bmyb) maps to mouse chromosome 2 and human chromosome 20q13.1
Noben-Trauth, K. [Jackson Lab., Bar Harbor, ME (United States)] [Jackson Lab., Bar Harbor, ME (United States); Copeland, N.G.; Gilbert, D.J. [ABL-Basic Research Program, Frederick, MD (United States)] [and others] [ABL-Basic Research Program, Frederick, MD (United States); and others
Mybl2 encodes a transcription factor that is though to play an important role in cell cycle progression. Here we report the chromosomal localization of Mybl2 in mouse and human. Using mouse Mybl2 cDNA clones as probes, we assigned Mybl2 in an interspecific backcross panel to distal Chromosome 2. Using human cDNA probes in combination with FISH analysis, we localized MYBL2 to chromosome 20q13.1, a region that is commonly deleted in myeloid disorders. Both chromosomal regions are highly homologous, and the map positions, therefore, confirm each other. However, our findings are in contrast to a previous report by Barletta et al. that placed the MYBL2 gene on human chromosome Xq.13. 21 refs., 2 figs.
The Construction of Transgenic and Gene Knockout/Knockin Mouse Models of Human Disease
Doyle, Alfred; McGarry, Michael P.; Lee, Nancy A.; Lee, James J.
The genetic and physiological similarities between mice and humans have focused considerable attention on rodents as potential models of human health and disease. Together with the wealth of resources, knowledge, and technologies surrounding the mouse as a model system, these similarities have propelled this species to the forefront of biomedical research. The advent of genomic manipulation has quickly led to the creation and use of genetically engineered mice as powerful tools for cutting edge studies of human disease research, including the discovery, refinement, and utility of many currently available therapeutic regimes. In particular, the creation of genetically modified mice as models of human disease has remarkably changed our ability to understand the molecular mechanisms and cellular pathways underlying disease states. Moreover, the mouse models resulting from gene transfer technologies have been important components correlating an individual�s gene expression profile to the development of disease pathologies. The objective of this review is to provide physician-scientists with an expansive historical and logistical overview of the creation of mouse models of human disease through gene transfer technologies. Our expectation is that this will facilitate on-going disease research studies and may initiate new areas of translational research leading to enhanced patient care. PMID:21800101
Conservation of exon scrambling in human and mouse
Hamilton, Monica L. (Monica Lauren)
Exon scrambling is a phenomenon in which the exons of an mRNA transcript are spliced in an order inconsistent with that of the genome. In this thesis, I present a computational analysis of scrambled exons in human and ...
Mouse-human immunoglobulin G1 chimeric antibodies with activities against Cryptococcus neoformans.
Zebedee, S L; Koduri, R K; Mukherjee, J; Mukherjee, S; Lee, S; Sauer, D F; Scharff, M D; Casadevall, A
Passive antibody administration is a potentially useful approach for the therapy of human Cryptococcus neoformans infections. To evaluate the efficacy of the human immunoglobulin G1 (IgG1) constant region against C. neoformans and to construct murine antibody derivatives with reduced immunogenicities and longer half-lives in humans, two mouse-human IgG1 chimeric antibodies were generated from the protective murine monoclonal antibodies 2D10 (IgM) and 18B7 (IgG1). The 2D10 mouse-human IgG1 chimeric antibody (ch2D10) had significantly lower binding affinity than its parent murine antibody (m2D10), presumably because of a loss of avidity contribution on switching from IgM to IgG. The 18B7 mouse-human IgG1 chimeric antibody (ch18B7) had higher affinity for cryptococcal polysaccharide antigen than its parent murine antibody (m18B7). ch18B7 and ch2D10 promoted phagocytosis of C. neoformans by primary human microglial cells and the murine J774.16 macrophage-like cell line. ch18B7 and m18B7 enhanced fungistatic or fungicidal activity of J774.16 cells and prolonged the survival of lethally infected mice. We conclude that the human IgG1 constant chain can be effective in mediating antifungal activity against C. neoformans. ch18B7 or similar antibodies are potential candidates for passive antibody therapy of human cryptococcosis. PMID:7979280
Radioimmunoscintigraphy using Technetium99m-labeled parental mouse and mouse-human chimeric antibodies to carcinoembryonic antigen in athymic nude mice bearing tumor
Yoshiharu Karube; Kentaro Katsuno; Jiro Takata; Kazuhisa Matsunaga; Masatora Haruno; Masahide Kuroki; Fumiko Arakawa; Yuji Matsuoka; Hidetoshi Kanda
Biodistribution and imaging characteristics of Tc-99m-labeled parental mouse and mouse-human chimeric antibodies to carcinoembryonic antigen (CEA), designated F11-39 and ChF11-39, respectively, were evaluated in athymic nude mice bearing the human CEA-producing gastric carcinoma (MKN-45) xenografts. Group F monoclonal antibodies such as F11-39 and ChF11-39 have been found to recognize the protein epitopes present on the domain B3 of the CEA
Development of a Mouse Model of Helicobacter pylori Infection that Mimics Human Disease
NASA Astrophysics Data System (ADS)
Marchetti, Marta; Arico, Beatrice; Burroni, Daniela; Figura, Natale; Rappuoli, Rino; Ghiara, Paolo
The human pathogen Helicobacter pylori is associated with gastritis, peptic ulcer disease, and gastric cancer. The pathogenesis of H. pylori infection in vivo was studied by adapting fresh clinical isolates of bacteria to colonize the stomachs of mice. A gastric pathology resembling human disease was observed in infections with cytotoxin-producing strains but not with noncytotoxic strains. Oral immunization with purified H. pylori antigens protected mice from bacterial infection. This mouse model will allow the development of therapeutic agents and vaccines against H. pylori infection in humans.
Transglutaminases expression in human supraspinatus tendon ruptures and in mouse tendons.
Oliva, Francesco; Zocchi, Loredana; Codispoti, Andrea; Candi, Eleonora; Celi, Monica; Melino, Gerry; Maffulli, Nicola; Tarantino, Umberto
The ethiopathogenesis of rotator cuff disease remains poorly understood. Many studies advocate the importance of extra cellular matrix for the homeostasis of connective tissue. Transglutaminase enzymes family has been studied in the context of connective tissue formation and stabilisation. Here, we investigated transglutaminases expression pattern in biopsies of normal and injured supraspinatus tendons of human shoulders and in the Achilles tendons of transglutaminase 2 knock-out and wild-type mice. Our results show that different transglutaminase family members are differentially expressed in human and mouse tendons, and that transglutaminase 2 is down-regulated at mRNA and protein levels upon human supraspinatus tendon ruptures. PMID:19146825
A cross-species genetic analysis identifies candidate genes for mouse anxiety and human bipolar disorder
Ashbrook, David G.; Williams, Robert W.; Lu, Lu; Hager, Reinmar
Bipolar disorder (BD) is a significant neuropsychiatric disorder with a lifetime prevalence of ~1%. To identify genetic variants underlying BD genome-wide association studies (GWAS) have been carried out. While many variants of small effect associated with BD have been identified few have yet been confirmed, partly because of the low power of GWAS due to multiple comparisons being made. Complementary mapping studies using murine models have identified genetic variants for behavioral traits linked to BD, often with high power, but these identified regions often contain too many genes for clear identification of candidate genes. In the current study we have aligned human BD GWAS results and mouse linkage studies to help define and evaluate candidate genes linked to BD, seeking to use the power of the mouse mapping with the precision of GWAS. We use quantitative trait mapping for open field test and elevated zero maze data in the largest mammalian model system, the BXD recombinant inbred mouse population, to identify genomic regions associated with these BD-like phenotypes. We then investigate these regions in whole genome data from the Psychiatric Genomics Consortium's bipolar disorder GWAS to identify candidate genes associated with BD. Finally we establish the biological relevance and pathways of these genes in a comprehensive systems genetics analysis. We identify four genes associated with both mouse anxiety and human BD. While TNR is a novel candidate for BD, we can confirm previously suggested associations with CMYA5, MCTP1, and RXRG. A cross-species, systems genetics analysis shows that MCTP1, RXRG, and TNR coexpress with genes linked to psychiatric disorders and identify the striatum as a potential site of action. CMYA5, MCTP1, RXRG, and TNR are associated with mouse anxiety and human BD. We hypothesize that MCTP1, RXRG, and TNR influence intercellular signaling in the striatum. PMID:26190982
Expanded conserved linkage group between human 16p13 and the Scid region of the mouse chromosome 16
Deng, Z.M.; Siciliano, M.J. [Univ. of Texas M.D. Anderson Cancer Center, Houston, TX (United States); Davisson, M.T. [Jackson Lab., Bar Harbor, ME (United States)] [and others
Knowledge of homologies between human and mouse chromosomes is essential for understanding chromosomal evolution and the development of experimental models for human disease. We have reported the identification of a conserved linkage group between human 16p13 and the centromeric portion of the mouse 16. Defining the extent of this linkage conservation has significant biomedical implications since that region of mouse genome contains the Scid mutation and the human 16p13 contains genes that are involved in DNA repair and certain types of human leukemia as well as other diseases such as Rubinstein-Taybi Syndrome. Here, this conserved linkage group has been defined and expanded. It now contains 5 genetic loci and spans more than 3 Mb in human and 23 cM in mouse. The 5 loci are PRM1,2 (protamine 1 and 2), NOP3 (a subclone of D16S237), GSPT1 (a gene involved in the regulation of G1 to S phase transition), MYH11 (a human smooth muscle myosin heavy chain gene) and MRP (multi-drug resistant-associated protein gene). Using a panel of human-rodent hybrids that are informative for different portions of human 16, we have established the following order on human 16p: telomere-NOP3-PRM1,2-GSPT1-(MYH11,MRP)-centromere. The genes were assigned to the mouse chromosome 16 by a mouse-Chinese hamster somatic cell hybrid panel informative for mouse chromosomes. Linkage analysis using backcross mice informative for the Scid mutation indicated the following order and genetic distance (in cM) in mouse: centromere-Nop3-11.7-Prm1-1.4-Gspt1-8.2-(Myh11,Mrp)-1.4-Scid-telomere.
Obesity genetics in mouse and human: back and forth, and back again
Yazdi, Fereshteh T.; Clee, Susanne M.
Obesity is a major public health concern. This condition results from a constant and complex interplay between predisposing genes and environmental stimuli. Current attempts to manage obesity have been moderately effective and a better understanding of the etiology of obesity is required for the development of more successful and personalized prevention and treatment options. To that effect, mouse models have been an essential tool in expanding our understanding of obesity, due to the availability of their complete genome sequence, genetically identified and defined strains, various tools for genetic manipulation and the accessibility of target tissues for obesity that are not easily attainable from humans. Our knowledge of monogenic obesity in humans greatly benefited from the mouse obesity genetics field. Genes underlying highly penetrant forms of monogenic obesity are part of the leptin-melanocortin pathway in the hypothalamus. Recently, hypothesis-generating genome-wide association studies for polygenic obesity traits in humans have led to the identification of 119 common gene variants with modest effect, most of them having an unknown function. These discoveries have led to novel animal models and have illuminated new biologic pathways. Integrated mouse-human genetic approaches have firmly established new obesity candidate genes. Innovative strategies recently developed by scientists are described in this review to accelerate the identification of causal genes and deepen our understanding of obesity etiology. An exhaustive dissection of the molecular roots of obesity may ultimately help to tackle the growing obesity epidemic worldwide. PMID:25825681
Comparison of epigenetic mediator expression and function in mouse and human embryonic blastomeres
Chavez, Shawn L.; McElroy, Sohyun L.; Bossert, Nancy L.; De Jonge, Christopher J.; Rodriguez, Maria Vera; Leong, Denise E.; Behr, Barry; Westphal, Lynn M.; Reijo Pera, Renee A.
A map of human embryo development that combines imaging, molecular, genetic and epigenetic data for comparisons to other species and across pathologies would be greatly beneficial for basic science and clinical applications. Here, we compared mRNA and protein expression of key mediators of DNA methylation and histone modifications between mouse and human embryos, embryos from fertile/infertile couples, and following growth factor supplementation. We observed that individual mouse and human embryos are characterized by similarities and distinct differences in DNA methylation and histone modification patterns especially at the single-cell level. In particular, while mouse embryos first exhibited sub-compartmentalization of different histone modifications between blastomeres at the morula stage and cell sub-populations in blastocysts, differential histone modification expression was detected between blastomeres earlier in human embryos at the four- to eight-cell stage. Likewise, differences in epigenetic mediator expression were also observed between embryos from fertile and infertile couples, which were largely equalized in response to growth factor supplementation, suggesting that select growth factors might prevent alterations in epigenetic profiles during prolonged embryo culture. Finally, we determined that reduced expression via morpholino technologies of a single histone-modifying enzyme, Rps6ka4/Msk2, resulted in cleavage-stage arrest as assessed by time-lapse imaging and was associated with aneuploidy generation. Taken together, data document differences in epigenetic patterns between species with implications for fertility and suggest functional roles for individual epigenetic factors during pre-implantation development. PMID:24821703
The human and mouse homologs of the yeat RAD52 gene: cDNA cloning, sequence analysis, assignment to human chromosome 12p12.2-p13, and mRNA expression in mouse tissues
Shen, Z.; Chen, D.J.; Denison, K. [Los Alamos National Laboratory, NM (United States)] [and others] [Los Alamos National Laboratory, NM (United States); and others
The yeast Saccharomyces cerevisiae RAD52 gene is involved in DNA double-strand break repair and mitotic/meiotic recombination. The N-terminal amino acid sequence of yeast S. cerevisiae, Schizosaccharomyces pombe, and Kluyveromyces lactis and chicken is highly conserved. Using the technology of mixed oligonucleotide primed amplification of cDNA (MOPAC), two mouse RAD52 homologous cDNA fragments were amplified and sequenced. Subsequently, we have cloned the cDNA of the human and mouse homologs of yeast RAD52 gene by screening cDNA libraries using the identified mouse cDNA fragments. Sequence analysis of cDNA derived amino acid revealed a highly conserved N-terminus among human, mouse, chicken, and yeast RAD52 genes. The human RAD52 gene was assigned to chromosome 12p12.2-p13 by fluorescence in situ hybridization, R-banding, and DNA analysis of somatic cell hybrids. Unlike chicken RAD52 and mouse RAD51, no significant difference in mouse RAD52 mRNA level was found among mouse heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis. In addition to an {approximately}1.9-kb RAD52 mRNA band that is present in all of the tested tissues, an extra mRNA species of {approximately}0.85 kb was detectable in mouse testis. 40 refs., 7 figs., 1 tab.
Using the BLT humanized mouse as a stem cell based gene therapy tumor model.
Vatakis, Dimitrios N; Bristol, Gregory C; Kim, Sohn G; Levin, Bernard; Liu, Wei; Radu, Caius G; Kitchen, Scott G; Zack, Jerome A
Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) (1, 2) or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) (3). Both models were mainly utilized for the study of HIV infection. One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant (4). The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency (5-8). We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC (7, 9). This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9). The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (http://jaxmice.jax.org/research/immunology/005557-housing.html). PMID:23271478
mMaspin: the mouse homolog of a human tumor suppressor gene inhibits mammary tumor invasion and motility.
Zhang, M.; Sheng, S.; Maass, N.; Sager, R.
BACKGROUND: The human maspin gene encodes a protein in the serine proteinase inhibitor (serpin) family with tumor-suppressing functions in cell culture and in nude mice. In order to examine the role of maspin in an intact mammal, we cloned and sequenced the cDNA of mouse maspin. The recombinant protein was produced and its activity in cell culture was assessed. MATERIALS AND METHODS: Mouse maspin (mMaspin) was cloned by screening a mouse mammary gland cDNA library with the human maspin cDNA probe. Northern blot analysis was used to examine the expression patterns in mouse tissues, mammary epithelial cells, and carcinomas. Recombinant mMaspin protein was produced in E. coli. Invasion and motility assays were used to assess the biological function of mMaspin. RESULTS: mMaspin is 89% homologous with human maspin at the amino acid level. Like its human homolog, mMaspin is expressed in normal mouse mammary epithelial cells and down-regulated in mouse breast tumor cell lines. The expression is altered at different developmental stages in mammary gland. Addition of the recombinant mMaspin protein to mouse tumor cells was shown to inhibit invasion in a dose-dependent manner. As with the human protein, recombinant mMaspin protein also inhibited mouse mammary tumor motility. Deletion in the putative mMaspin reactive site loop (RSL) region resulted in the loss of its inhibitory functions. CONCLUSIONS: mMaspin is the mouse homolog of a human tumor suppressor gene. The expression of mMaspin is down-regulated in tumor cells and is altered at different developmental stages of mammary gland. mMaspin has inhibitory properties similar to those of human maspin in cell culture, suggesting that the homologous proteins play similar physiological roles in vivo. Images FIG. 3 FIG. 4 FIG. 5 FIG. 7 PMID:9132279
Genomic analysis of mouse tumorigenesis
Tam, Mandy Chi-Mun
The availability of the human and mouse genome sequences has spurred a growing interest in analyzing mouse models of human cancer using genomic techniques. Comparative genomic studies on mouse and human tumors can be ...
Human and Mouse CD137 Have Predominantly Different Binding CRDs to Their Respective Ligands
Yi, Ling; Zhao, Yanlin; Wang, Xiaojue; Dai, Min; Hellstr�m, Karl Erik; Hellstr�m, Ingegerd; Zhang, Hongtao
Monoclonal antibodies (mAbs) to CD137 (a.k.a. 4-1BB) have anti-tumor efficacy in several animal models and have entered clinical trials in patients with advanced cancer. Importantly, anti-CD137 mAbs can also ameliorate autoimmunity in preclinical models. As an approach to better understand the action of agonistic and antagonistic anti-CD137 mAbs we have mapped the binding region of the CD137 ligand (CD137L) to human and mouse CD137. By investigating the binding of CD137L to cysteine rich domain II (CRDII )and CRDIII of CD137, we found that the binding interface was limited and differed between the two species in that mouse CD137L mainly combined with CRDII and human CD137L mainly combined with CRDIII. PMID:24466035
Genetic conflict reflected in tissue-specific maps of genomic imprinting in human and mouse.
Babak, Tomas; DeVeale, Brian; Tsang, Emily K; Zhou, Yiqi; Li, Xin; Smith, Kevin S; Kukurba, Kim R; Zhang, Rui; Li, Jin Billy; van der Kooy, Derek; Montgomery, Stephen B; Fraser, Hunter B
Genomic imprinting is an epigenetic process that restricts gene expression to either the maternally or paternally inherited allele. Many theories have been proposed to explain its evolutionary origin, but understanding has been limited by a paucity of data mapping the breadth and dynamics of imprinting within any organism. We generated an atlas of imprinting spanning 33 mouse and 45 human developmental stages and tissues. Nearly all imprinted genes were imprinted in early development and either retained their parent-of-origin expression in adults or lost it completely. Consistent with an evolutionary signature of parental conflict, imprinted genes were enriched for coexpressed pairs of maternally and paternally expressed genes, showed accelerated expression divergence between human and mouse, and were more highly expressed than their non-imprinted orthologs in other species. Our approach demonstrates a general framework for the discovery of imprinting in any species and sheds light on the causes and consequences of genomic imprinting in mammals. PMID:25848752
Visualization of plasmid delivery to keratinocytes in mouse and human epidermis
Gonz�lez-Gonz�lez, Emilio; Kim, Yeu-Chun; Speaker, Tycho J.; Hickerson, Robyn P.; Spitler, Ryan; Birchall, James C.; Lara, Maria Fernanda; Hu, Rong-hua; Liang, Yanhua; Kirkiles-Smith, Nancy; Prausnitz, Mark R.; Milstone, Leonard M.; Contag, Christopher H.; Kaspar, Roger L.
The accessibility of skin makes it an ideal target organ for nucleic acid-based therapeutics; however, effective patient-friendly delivery remains a major obstacle to clinical utility. A variety of limited and inefficient methods of delivering nucleic acids to keratinocytes have been demonstrated; further advances will require well-characterized reagents, rapid noninvasive assays of delivery, and well-developed skin model systems. Using intravital fluorescence and bioluminescence imaging and a standard set of reporter plasmids we demonstrate transfection of cells in mouse and human xenograft skin using intradermal injection and two microneedle array delivery systems. Reporter gene expression could be detected in individual keratinocytes, in real-time, in both mouse skin as well as human skin xenografts. These studies revealed that non-invasive intravital imaging can be used as a guide for developing gene delivery tools, establishing a benchmark for comparative testing of nucleic acid skin delivery technologies. PMID:22355673
Plasmacytoma-associated neuronal glycoprotein, Pang, maps to mouse chromosome 6 and human chromosome 3
Mock, B.A.; McBride, O.W.; Kozak, C.A. [NIH, Bethesda, MD (United States)] [and others] [NIH, Bethesda, MD (United States); and others
A new member of the immunoglobulin/fibronectin superfamily of adhesion molecules, Pang (plasmacytoma-associated neuronal glycoprotein), was recently isolated from a plasmacytoma. In previous studies, Pang was found to be normally expressed in the brain and ectopically activated by intracisternal A-type particle long terminal repeats in plasmacytomas. In this study, Pang was initially mapped to mouse Chr 6 by somatic cell hybrid analysis and further positioned on the chromosome between Wnt7a and Pcp1. Southern blot analysis of human-rodent somatic cell hybrids together with predictions from the mouse map location indicate that human PANG is located at 3p26. 13 refs., 1 fig., 1 tab.
Generation of L-cells in mouse and human small intestine organoids
Petersen, Natalia; Reimann, Frank; Bartfeld, Sina; Farin, Henner F.; Ringnalda, Femke C.; Vries, Robert G. J.; van den Brink, Stieneke; Clevers, Hans; Gribble, Fiona M.; de Koning, Eelco J. P.
Upon a nutrient challenge, L-cells produce glucagon-like peptide 1 (GLP-1), a powerful stimulant of insulin release. Strategies to augment endogenous GLP-1 production include promoting L-cell differentiation and increasing L-cell number. Here we present a novel in vitro platform to generate functional L-cells from 3D cultures of mouse and human intestinal crypts. We show that short-chain fatty acids (SCFAs) selectively increase the number of L-cells resulting in an elevation of GLP-1 release. This is accompanied by up-regulation of transcription factors, associated with the endocrine lineage of intestinal stem cell development. Thus, our platform allows us to study and modulate the development of L-cells in mouse and human crypts as a potential basis for novel therapeutic strategies in type 2 diabetes. PMID:24130334
The Consensus Coding Sequence (Ccds) Project: Identifying a Common Protein-Coding Gene Set for the Human and Mouse Genomes
Kellis, Manolis
Effective use of the human and mouse genomes requires reliable identification of genes and their products. Although multiple public resources provide annotation, different methods are used that can result in similar but ...
Noise in a laboratory animal facility from the human and mouse perspectives.
Reynolds, Randall P; Kinard, Will L; Degraff, Jesse J; Leverage, Ned; Norton, John N
The current study was performed to understand the level of sound produced by ventilated racks, animal transfer stations, and construction equipment that mice in ventilated cages hear relative to what humans would hear in the same environment. Although the ventilated rack and animal transfer station both produced sound pressure levels above the ambient level within the human hearing range, the sound pressure levels within the mouse hearing range did not increase above ambient noise from either noise source. When various types of construction equipment were used 3 ft from the ventilated rack, the sound pressure level within the mouse hearing range was increased but to a lesser degree for each implement than were the sound pressure levels within the human hearing range. At more distant locations within the animal facility, sound pressure levels from the large jackhammer within the mouse hearing range decreased much more rapidly than did those in the human hearing range, indicating that less of the sound is perceived by mice than by humans. The relatively high proportion of low-frequency sound produced by the shot blaster, used without the metal shot that it normally uses to clean concrete, increased the sound pressure level above the ambient level for humans but did not increase sound pressure levels above ambient noise for mice at locations greater than 3 ft from inside of the cage, where sound was measured. This study demonstrates that sound clearly audible to humans in the animal facility may be perceived to a lesser degree or not at all by mice, because of the frequency content of the sound. PMID:20858361
Metabolism of the anti-tuberculosis drug ethionamide by mouse and human FMO1, FMO2 and FMO3 and mouse and human lung microsomes
Henderson, Marilyn C.; Siddens, Lisbeth K. [Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 97331-7301 (United States); Morre, Jeffrey T. [Environmental Health Sciences Center, Oregon State University, Corvallis, OR 97331-7302 (United States); Krueger, Sharon K. [Linus Pauling Institute, Oregon State University, Corvallis, OR 97331-6512 (United States); Williams, David E. [Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 97331-7301 (United States); Environmental Health Sciences Center, Oregon State University, Corvallis, OR 97331-7302 (United States); Linus Pauling Institute, Oregon State University, Corvallis, OR 97331-6512 (United States)], E-mail: email@example.com
Tuberculosis (TB) results from infection with Mycobacterium tuberculosis and remains endemic throughout the world with one-third of the world's population infected. The prevalence of multi-drug resistant strains necessitates the use of more toxic second-line drugs such as ethionamide (ETA), a pro-drug requiring bioactivation to exert toxicity. M. tuberculosis possesses a flavin monooxygenase (EtaA) that oxygenates ETA first to the sulfoxide and then to 2-ethyl-4-amidopyridine, presumably through a second oxygenation involving sulfinic acid. ETA is also a substrate for mammalian flavin-containing monooxygenases (FMOs). We examined activity of expressed human and mouse FMOs toward ETA, as well as liver and lung microsomes. All FMOs converted ETA to the S-oxide (ETASO), the first step in bioactivation. Compared to M. tuberculosis, the second S-oxygenation to the sulfinic acid is slow. Mouse liver and lung microsomes, as well as human lung microsomes from an individual expressing active FMO, oxygenated ETA in the same manner as expressed FMOs, confirming this reaction functions in the major target organs for therapeutics (lung) and toxicity (liver). Inhibition by thiourea, and lack of inhibition by SKF-525A, confirm ETASO formation is primarily via FMO, particularly in lung. ETASO production was attenuated in a concentration-dependent manner by glutathione. FMO3 in human liver may contribute to the toxicity and/or affect efficacy of ETA administration. Additionally, there may be therapeutic implications of efficacy and toxicity in human lung based on the FMO2 genetic polymorphism, though further studies are needed to confirm that suggestion.
Organoid models of human and mouse ductal pancreatic cancer.
Boj, Sylvia F; Hwang, Chang-Il; Baker, Lindsey A; Chio, Iok In Christine; Engle, Dannielle D; Corbo, Vincenzo; Jager, Myrthe; Ponz-Sarvise, Mariano; Tiriac, Herv�; Spector, Mona S; Gracanin, Ana; Oni, Tobiloba; Yu, Kenneth H; van Boxtel, Ruben; Huch, Meritxell; Rivera, Keith D; Wilson, John P; Feigin, Michael E; �hlund, Daniel; Handly-Santana, Abram; Ardito-Abraham, Christine M; Ludwig, Michael; Elyada, Ela; Alagesan, Brinda; Biffi, Giulia; Yordanov, Georgi N; Delcuze, Bethany; Creighton, Brianna; Wright, Kevin; Park, Youngkyu; Morsink, Folkert H M; Molenaar, I Quintus; Borel Rinkes, Inne H; Cuppen, Edwin; Hao, Yuan; Jin, Ying; Nijman, Isaac J; Iacobuzio-Donahue, Christine; Leach, Steven D; Pappin, Darryl J; Hammell, Molly; Klimstra, David S; Basturk, Olca; Hruban, Ralph H; Offerhaus, George Johan; Vries, Robert G J; Clevers, Hans; Tuveson, David A
Pancreatic cancer is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable methods to identify and interrogate pathways involved in pancreatic tumorigenesis are urgently needed. We established organoid models from normal and neoplastic murine and human pancreas tissues. Pancreatic organoids can be rapidly generated from resected tumors and biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Orthotopically transplanted neoplastic organoids recapitulate the full spectrum of tumor development by forming early-grade neoplasms that progress to locally invasive and metastatic carcinomas. Due to their ability to be genetically manipulated, organoids are a platform to probe genetic cooperation. Comprehensive transcriptional and proteomic analyses of murine pancreatic organoids revealed genes and pathways altered during disease progression. The confirmation of many of these protein changes in human tissues demonstrates that organoids are a facile model system to discover characteristics of this deadly malignancy. PMID:25557080
Reprogramming of mouse and human somatic cells by high-performance engineered factors
Yang Wang; Jiekai Chen; Jia-Lei Hu; Xi-Xiao Wei; Dajiang Qin; Juan Gao; Lei Zhang; Jing Jiang; Jin-Song Li; Jing Liu; Ke-Yu Lai; Xia Kuang; Jian Zhang; Duanqing Pei; Guo-Liang Xu
Reprogramming somatic cells to become induced pluripotent stem cells (iPSCs) by using defined factors represents an important breakthrough in biology and medicine, yet remains inefficient and poorly understood. We therefore devised synthetic factors by fusing the VP16 transactivation domain to OCT4 (also known as Pou5f1), NANOG and SOX2, respectively. These synthetic factors could reprogramme both mouse and human fibroblasts with
Bioelectric Properties of Chloride Channels in Human, Pig, Ferret and Mouse Airway Epithelia
Xiaoming Liu; Meihui Luo; Liang Zhang; Wei Ding; Ziying Yan; John F. Engelhardt
The development of effective therapies for cystic fibrosis (CF) re- quires animal models that can appropriately reproduce the human disease phenotype. CF mouse models have demonstrated cAMP- inducible, non-CF transmembrane conductance regulator (non- CFTR) chloride transport in conducting airway epithelia, and this property is thought to be responsible for the lack of a spontaneous CF-like phenotype in the lung. Thus,
Characterization of the human PAP1 gene and its homologue possible involvement in mouse embryonic development
Kun-Xian Shu; Li-Xiang Wu; Yong-Fang Xie; Jin-Feng Zhao; Yi-Long Liang; Biao Li
We have identified PAP1 gene, a novel member of the immunoglobulin superfamily (IGSF) from U251-pTet-p53 cell line, which carried a wild-type p53 transgene. The gene has been localised to chromosome 16p12-13. Alignment of the predicted protein sequence for Human, Pan troglodytes, Canis, Mus musculus and Gallus gallus revealed it was highly conserved. Its homologue, IGSF6, possible involves in mouse embryonic
Functional validation of a human CAPN5 exome variant by lentiviral transduction into mouse retina
Wert, Katherine J.; Skeie, Jessica M.; Bassuk, Alexander G.; Olivier, Alicia K.; Tsang, Stephen H.; Mahajan, Vinit B.
Exome sequencing indicated that the gene encoding the calpain-5 protease, CAPN5, is the likely cause of retinal degeneration and autoimmune uveitis in human patients with autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV, OMIM #193235). To explore the mechanism of ADNIV, a human CAPN5 disease allele was expressed in mouse retinas with a lentiviral vector created to express either the wild-type human (h) CAPN5 or the ADNIV mutant hCAPN5-R243L allele under a rhodopsin promoter with tandem green fluorescent protein (GFP) expression. Vectors were injected into the subretinal space of perinatal mice. Mouse phenotypes were analyzed using electroretinography, histology and inflammatory gene expression profiling. Mouse calpain-5 showed high homology to its human ortholog with >98% sequence identity that includes the ADNIV mutant residue. Calpain-5 protein was expressed in the inner and outer segments of the photoreceptors and in the outer plexiform layer. Expression of the hCAPN5-R243L allele caused loss of the electroretinogram b-wave, photoreceptor degeneration and induction of immune cell infiltration and inflammatory genes in the retina, recapitulating major features of the ADNIV phenotype. Intraocular neovascularization and fibrosis were not observed during the study period. Our study shows that expression of the hCAPN5-R243L disease allele elicits an ADNIV-like disease in mice. It further suggests that ADNIV is due to CAPN5 gain-of-function rather than haploinsufficiency, and retinal expression may be sufficient to generate an autoimmune response. Genetic models of ADNIV in the mouse can be used to explore protease mechanisms in retinal degeneration and inflammation as well as preclinical therapeutic testing. PMID:24381307
Gene structure of human and mouse methylenetetrahydrofolate reductase (MTHFR)
Philippe Goyette; Aditya Pai; Renate Milos; Phyllis Frosst; Pamela Tran; Zhoutao Chen; Manuel Chan; Rima Rozen
.�� Methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate,\\u000a a co-substrate for homocysteine remethylation to methionine. A human cDNA for MTHFR, 2.2 kb in length, has been expressed\\u000a and shown to result in a catalytically active enzyme of approximately 70 kDa. Fifteen mutations have been identified in the\\u000a MTHFR gene: 14 rare mutations associated with severe enzymatic deficiency
Isolation of myofibroblasts from mouse and human esophagus.
Gargus, Matthew; Niu, Chao; Shaker, Anisa
Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express ?-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders. PMID:25650889
Retinoid metabolism is altered in human and mouse cicatricial alopecia
Everts, Helen B.; Silva, Kathleen A.; Montgomery, Shalise; Suo, Liye; Menser, Monica; Valet, Amy S.; King, Lloyd E.; Ong, David E; Sundberg, John P.
C57BL/6 mice develop dermatitis and scarring alopecia resembling human cicatricial alopecias (CA), particularly the central centrifugal cicatricial alopecia (CCCA) type. To evaluate the role of retinoids in CA, expression of retinoid metabolism components were examined in these mice with mild, moderate, or severe CA compared to hair cycle matched mice with no disease. Two feeding studies were performed with dams fed either NIH 31 diet (study 1) or AIN93G diet (study 2). Adult mice were fed AIN93M diet with 4 (recommended), 28, or 56 IU vitamin A/g diet. Feeding the AIN93M diet to adults increased CA frequency over NIH 31 fed mice. Increased follicular dystrophy was seen in study 1 and increased dermal scars in study 2 in mice fed the 28 IU diet. These results indicate that retinoid metabolism is altered in CA in C57BL/6J mice that require precise levels of dietary vitamin A. Human patients with CCCA, pseudopelade (end stage scarring), and controls with no alopecia were also studied. Many retinoid metabolism proteins were increased in mild CCCA, but were undetectable in pseudopelade. Studies to determine if these dietary alterations in retinoid metabolism seen in C57BL/6J mice are also involved in different types of human CA are needed. PMID:23096705
Chimeric antibody with human constant regions and mouse variable regions directed against carcinoma-associated antigen 17-1A
L. K. Sun; P. Curtis; E. Rakowicz-Szulczynska; J. Ghrayeb; N. Chang; S. L. Morrison; H. Koprowski
The authors have cloned the genomic DNA fragments encoding the heavy and light chain variable regions of monoclonal antibody 17-1A, and they have inserted them into mammalian expression vectors containing genomic DNA segments encoding human ..gamma..3 and kappa constant regions. The transfer of these expression vectors containing mouse-human chimeric immunoglobulin genes into Sp2\\/0 mouse myeloma cells resulted in the production
Chimeric Antibody with Human Constant Regions and Mouse Variable Regions Directed against Carcinoma-Associated Antigen 17-1A
Lee K. Sun; Peter Curtis; Eva Rakowicz-Szulczynska; John Ghrayeb; Nancy Chang; Sherie L. Morrison; Hilary Koprowski
We have cloned the genomic DNA fragments encoding the heavy and light chain variable regions of monoclonal antibody 17-1A, and we have inserted them into mammalian expression vectors containing genomic DNA segments encoding human gamma 3 and kappa constant regions. The transfer of these expression vectors containing mouse-human chimeric immunoglobulin genes into Sp2\\/0 mouse myeloma cells resulted in the production
Expression of Human Epidermal Growth Factor Precursor cDNA in Transfected Mouse NIH 3T3 Cells
Barbara Mroczkowski; Martha Reich; Jonathan Whittaker; Graeme I. Bell; Stanley Cohen
Stable cell lines expressing the human epidermal growth factor (EGF) precursor have been prepared by transfection of mouse NIH 3T3 cells with a bovine papillomavirus-based vector in which the human kidney EGF precursor cDNA has been placed under the control of the inducible mouse metallothionein I promoter. Synthesis of the EGF precursor can be induced by culturing the cells in
FAAH genetic variation enhances fronto-amygdala function in mouse and human.
Dincheva, Iva; Drysdale, Andrew T; Hartley, Catherine A; Johnson, David C; Jing, Deqiang; King, Elizabeth C; Ra, Stephen; Gray, J Megan; Yang, Ruirong; DeGruccio, Ann Marie; Huang, Chienchun; Cravatt, Benjamin F; Glatt, Charles E; Hill, Matthew N; Casey, B J; Lee, Francis S
Cross-species studies enable rapid translational discovery and produce the broadest impact when both mechanism and phenotype are consistent across organisms. We developed a knock-in mouse that biologically recapitulates a common human mutation in the gene for fatty acid amide hydrolase (FAAH) (C385A; rs324420), the primary catabolic enzyme for the endocannabinoid anandamide. This common polymorphism impacts the expression and activity of FAAH, thereby increasing anandamide levels. Here, we show that the genetic knock-in mouse and human variant allele carriers exhibit parallel alterations in biochemisty, neurocircuitry and behaviour. Specifically, there is reduced FAAH expression associated with the variant allele that selectively enhances fronto-amygdala connectivity and fear extinction learning, and decreases anxiety-like behaviours. These results suggest a gain of function in fear regulation and may indicate for whom and for what anxiety symptoms FAAH inhibitors or exposure-based therapies will be most efficacious, bridging an important translational gap between the mouse and human. PMID:25731744
Identification of the human homolog of the imprinted mouse Air non-coding RNA
Yotova, Iveta Y.; Vlatkovic, Irena M.; Pauler, Florian M.; Warczok, Katarzyna E.; Ambros, Peter F.; Oshimura, Mitsuo; Theussl, Hans-Christian; Gessler, Manfred; Wagner, Erwin F.; Barlow, Denise P.
Genomic imprinting is widely conserved amongst placental mammals. Imprinted expression of IGF2R, however, differs between mice and humans. In mice, Igf2r imprinted expression is seen in all fetal and adult tissues. In humans, adult tissues lack IGF2R imprinted expression, but it is found in fetal tissues and Wilms' tumors where it is polymorphic and only seen in a small proportion of tested samples. Mouse Igf2r imprinted expression is controlled by the Air (Airn) ncRNA whose promoter lies in an intronic maternally-methylated CpG island. The human IGF2R gene carries a homologous intronic maternally-methylated CpG island of unknown function. Here, we use transfection and transgenic studies to show that the human IGF2R intronic CpG island is a ncRNA promoter. We also identify the same ncRNA at the endogenous human locus in 16�40% of Wilms' tumors. Thus, the human IGF2R gene shows evolutionary conservation of key features that control imprinted expression in the mouse. PMID:18789384
The mouse/human cross-species heterodimer of leucine-rich repeat kinase 2: possible significance in the transgenic model mouse of Parkinson's disease.
Miyajima, T; Ohta, E; Kawada, H; Maekawa, T; Obata, F
Leucine-rich repeat kinase (LRRK2) is the causal molecule of autosomal dominant Parkinson's disease (PD). We previously reported that intracellular degradation of wild-type (WT) LRRK2 is promoted by formation of heterodimers with the I2020T mutant LRRK2. In the present study, we investigated whether this is also the case for mouse/human cross-species heterodimers, which could be formed in transgenic mice. First, by co-transfection and immunoprecipitation, we identified the cross-species heterodimer of mouse LRRK2 and human LRRK2. Next, we found that the protein level of mouse LRRK2 decreased when co-transfected with human I2020T LRRK2, but not with human WT LRRK2. These results suggested that degradation of mouse LRRK2 was promoted by formation of a cross-species heterodimer with the mutant LRRK2. In I2020T LRRK2-transgenic mice, the lower protein level of brain LRRK2 in comparison with control mice, together with higher expression of the mRNA, suggested that endogenous LRRK2 was degraded by formation of cross-species heterodimers. Our results suggest a new concept of cross-species dimer/oligomer formation in transgenic disease-model mice. PMID:25562633
Expression of human and mouse adenine nucleotide translocase (ANT) isoform genes in adipogenesis.
Gavald�-Navarro, Aleix; Domingo, Pere; Vi�as, Octavi; Mampel, Teresa
Adenine nucleotide translocases (ANTs) are mitochondrial proteins encoded by nuclear DNA that catalyze the exchange of ATP generated in the mitochondria for ADP produced in cytosol. There are four ANT isoforms in humans (hANT1-4) and three in mice (mANT1, mANT2 and mANT4), all encoded by distinct genes. The aim of this study was to quantify expression of ANT isoform genes during the adipogenesis of mouse 3T3-L1 and human Simpson-Golabi-Behmel syndrome (SGBS)-derived preadipocytes. We also studied the effects of the adipogenesis regulators, insulin and rosiglitazone, on ANT isoform expression in differentiated adipocytes and examined the expression of ANT isoforms in subcutaneous and visceral white adipose tissue (WAT) from mice and humans. We found that adipogenesis was associated with an increase in the expression of ANT isoforms, specifically mANT2 in mouse 3T3-L1 cells and hANT3 in human SGBS cells. These changes could be involved in the increases in oxidative metabolism and decreases in lactate production observed during differentiation. Insulin and rosiglitazone induced mANT2 gene expression in mature 3T3-L1 cells and hANT2 and hANT3 gene expression in SGBS adipocytes. Furthermore, human WAT expressed greater amounts of hANT3 than hANT2, and the expression of both of these isoforms was greater in subcutaneous WAT than in visceral WAT. Finally, inhibition of ANT activity by atractyloside or bongkrekic acid impaired proper adipocyte differentiation. These results suggest that changes in the expression of ANT isoforms may be involved in adipogenesis in both human and mouse WAT. PMID:25817039
Functional Integration of Human Neural Precursor Cells in Mouse Cortex
Zhou, Fu-Wen; Fortin, Jeff M.; Chen, Huan-Xin; Martinez-Diaz, Hildabelis; Chang, Lung-Ji; Reynolds, Brent A.; Roper, Steven N.
This study investigates the electrophysiological properties and functional integration of different phenotypes of transplanted human neural precursor cells (hNPCs) in immunodeficient NSG mice. Postnatal day 2 mice received unilateral injections of 100,000 GFP+ hNPCs into the right parietal cortex. Eight weeks after transplantation, 1.21% of transplanted hNPCs survived. In these hNPCs, parvalbumin (PV)-, calretinin (CR)-, somatostatin (SS)-positive inhibitory interneurons and excitatory pyramidal neurons were confirmed electrophysiologically and histologically. All GFP+ hNPCs were immunoreactive with anti-human specific nuclear protein. The proportions of PV-, CR-, and SS-positive cells among GFP+ cells were 35.5%, 15.7%, and 17.1%, respectively; around 15% of GFP+ cells were identified as pyramidal neurons. Those electrophysiologically and histological identified GFP+ hNPCs were shown to fire action potentials with the appropriate firing patterns for different classes of neurons and to display spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs). The amplitude, frequency and kinetic properties of sEPSCs and sIPSCs in different types of hNPCs were comparable to host cells of the same type. In conclusion, GFP+ hNPCs produce neurons that are competent to integrate functionally into host neocortical neuronal networks. This provides promising data on the potential for hNPCs to serve as therapeutic agents in neurological diseases with abnormal neuronal circuitry such as epilepsy. PMID:25763840
Characterization of multidrug transporter-mediated efflux of avermectins in human and mouse neuroblastoma cell lines.
Dalzell, Abigail M; Mistry, Pratibha; Wright, Jayne; Williams, Faith M; Brown, Colin D A
ABC transporters play an important role in the disposition of avermectins in several animal species. In this study the interactions of three key avermectins, abamectin, emamectin and ivermectin, with human and mouse homologues of MDR1 (ABCB1/Abcb1a) and MRP (ABCC/Abcc), transporters endogenously expressed by human SH-SY5Y and mouse N2a neuroblastoma cells were investigated. In both cell lines, retention of the fluorescent dye H33342 was found to be significantly increased in the presence of avermectins and cyclosporin A. These effects were shown to be unresponsive to the BCRP inhibitor Ko-143 and therefore MDR1/Mdr1-dependent. Avermectins inhibited MDR1/Mdr1a-mediated H33342 dye efflux, with apparent Ki values of 0.24�0.08 and 0.18�0.02?M (ivermectin); 0.60�0.07 and 0.56�0.02?M (emamectin) and 0.95�0.08 and 0.77�0.25?M (abamectin) in SH-SY5Y and N2a cells, respectively. There were some apparent affinity differences for MDR1 and Mdr1a within each cell line (affinity for ivermectin>emamectin?abamectin, P<0.05 by One-Way ANOVA), but importantly, the Ki values for individual avermectins for human MDR1 or mouse Mdr1a were not significantly different. MK571-sensitive retention of GSMF confirmed the expression of MRP/Mrp efflux transporters in both cell lines. Avermectins inhibited MRP/Mrp-mediated dye efflux with IC50 values of 1.58�0.51 and 1.94�0.72?M (ivermectin); 1.87�0.57 and 2.74�1.01?M (emamectin) and 2.25�0.01 and 1.68�0.63?M (abamectin) in SH-SY5Y and N2a cells, respectively. There were no significant differences in IC50 values between individual avermectins or between human MRP and mouse Mrp. Kinetic data for endogenous human MDR1/MRP isoforms in SH-SY5Y cells and mouse Mdr1a/b/Mrp isoforms in N2a cells are comparable for the selected avermectins. All are effluxed at concentrations well above 0.05-0.1?M ivermectin detected in plasma (Ottesen and Campbell, 1994; Ottesen and Campbell, 1994) This is an important finding in the light of toxicity seen in the Mdr1-deficient animal models CF-1 mice, Mdr1ab (-/-) double knockout mice and Collie dogs. We also confirm MRP/Mrp-mediated avermectin transport in both N2a and SH-SY5Y cell lines. PMID:25865432
The PanK2 Genes of Mouse and Human Specify Proteins with DistinctSubcellular Locations
Leonardi, Roberta; Zhang, Yong-Mei; Lydikis, Athanasios; Stevens,Robert D.; Ilkayeva, Olga R.; Wenner, Brett R.; Bain, James R.; Newgard,Christopher B.; Rock, Charles O.; Jackowski, Suzanne
Coenzyme A (CoA) biosynthesis is initiated by pantothenatekinase (PanK) and CoA levels are controlled through differentialexpression and feedback regulation of PanK isoforms. PanK2 is amitochondrial protein in humans, but comparative genomics revealed thatacquisition of a mitochondrial targeting signal was limited to primates.Human and mouse PanK2 possessed similar biochemical properties, withinhibition by acetylCoA and activation by palmitoylcarnitine. Mouse PanK2localized in the cytosol, and the expression of PanK2 was higher in humanbrain compared to mouse brain. Differences in expression and subcellularlocalization should be considered in developing a mouse model for humanPanK2 deficiency. (c) 2007 Federation of European Biochemical Societies.Published by Elsevier B.V.
High-performance liquid chromatography analysis of a novel small-molecule, anti-cancer drug, Palomid 529, in human and mouse plasma and in mouse tissue homogenates
Fan Lin; David Sherris; Jos H. Beijnen; Olaf Van Tellingen
Palomid 529 (8-(1-Hydroxy-ethyl)-2-methoxy-3-(4-methoxy-benzyloxy)-benzo[c]chromen-6-one), is a novel non-steroidal small-molecule drug, which inhibits both mTORC1 and mTORC2 assembly, and elicits both anti-angiogenic and direct anti-tumor effects in vivo. We have developed and validated a sensitive and selective method for the quantification of Palomid 529 in human and mouse plasma and in a range of mouse tissue samples. Sample pretreatment involved liquid�liquid extraction
Quantitation of fixative-induced morphologic and antigenic variation in mouse and human breast cancers
Cardiff, Robert D; Hubbard, Neil E; Engelberg, Jesse A; Munn, Robert J; Miller, Claramae H; Walls, Judith E; Chen, Jane Q; Vel�squez-Garc�a, H�ctor A; Galvez, Jose J; Bell, Katie J; Beckett, Laurel A; Li, Yue-Ju; Borowsky, Alexander D
Quantitative Image Analysis (QIA) of digitized whole slide images for morphometric parameters and immunohistochemistry of breast cancer antigens was used to evaluate the technical reproducibility, biological variability, and intratumoral heterogeneity in three transplantable mouse mammary tumor models of human breast cancer. The relative preservation of structure and immunogenicity of the three mouse models and three human breast cancers was also compared when fixed with representatives of four distinct classes of fixatives. The three mouse mammary tumor cell models were an ER + /PR + model (SSM2), a Her2 + model (NDL), and a triple negative model (MET1). The four breast cancer antigens were ER, PR, Her2, and Ki67. The fixatives included examples of (1) strong cross-linkers, (2) weak cross-linkers, (3) coagulants, and (4) combination fixatives. Each parameter was quantitatively analyzed using modified Aperio Technologies ImageScope algorithms. Careful pre-analytical adjustments to the algorithms were required to provide accurate results. The QIA permitted rigorous statistical analysis of results and grading by rank order. The analyses suggested excellent technical reproducibility and confirmed biological heterogeneity within each tumor. The strong cross-linker fixatives, such as formalin, consistently ranked higher than weak cross-linker, coagulant and combination fixatives in both the morphometric and immunohistochemical parameters. PMID:23399853
Quantitation of fixative-induced morphologic and antigenic variation in mouse and human breast cancers.
Cardiff, Robert D; Hubbard, Neil E; Engelberg, Jesse A; Munn, Robert J; Miller, Claramae H; Walls, Judith E; Chen, Jane Q; Vel�squez-Garc�a, H�ctor A; Galvez, Jose J; Bell, Katie J; Beckett, Laurel A; Li, Yue-Ju; Borowsky, Alexander D
Quantitative Image Analysis (QIA) of digitized whole slide images for morphometric parameters and immunohistochemistry of breast cancer antigens was used to evaluate the technical reproducibility, biological variability, and intratumoral heterogeneity in three transplantable mouse mammary tumor models of human breast cancer. The relative preservation of structure and immunogenicity of the three mouse models and three human breast cancers was also compared when fixed with representatives of four distinct classes of fixatives. The three mouse mammary tumor cell models were an ER+/PR+ model (SSM2), a Her2+ model (NDL), and a triple negative model (MET1). The four breast cancer antigens were ER, PR, Her2, and Ki67. The fixatives included examples of (1) strong cross-linkers, (2) weak cross-linkers, (3) coagulants, and (4) combination fixatives. Each parameter was quantitatively analyzed using modified Aperio Technologies ImageScope algorithms. Careful pre-analytical adjustments to the algorithms were required to provide accurate results. The QIA permitted rigorous statistical analysis of results and grading by rank order. The analyses suggested excellent technical reproducibility and confirmed biological heterogeneity within each tumor. The strong cross-linker fixatives, such as formalin, consistently ranked higher than weak cross-linker, coagulant and combination fixatives in both the morphometric and immunohistochemical parameters. PMID:23399853
Comparative mapping of 50 human chromosome 9 loci in the laboratory mouse.
Pilz, A; Woodward, K; Povey, S; Abbott, C
We have set out to produce a comprehensive comparative map between human chromosome 9 (HSA9) and the laboratory mouse. The mouse homologues of 50 loci that were known to map to HSA9 were mapped by interspecific backcross linkage analysis. Ten loci from the short arm of HSA9 were mapped, and 40 from HSA9q, with 24 markers coming from the HSA9q33-q34 region--a part of the chromosome known to be very gene rich. Fifteen new assignments have been made--Ak3, Ctsl, Cntfr, C8g, D2H9S46E, Eng, Gcnt1, Irebp, Pappa, Ptgds, Snf212, Tal2, Tmod, Vav2, and Vldlr, the human homologues of which all map to HSA9. In addition, the assignment of Snf212 and Vldlr to MMU19 has defined a new region of synteny between the proximal portion of the short arm of HSA9 and the mouse. PMID:7774911
Cryptic Translocation Identification in Human and Mouse using Several Telomeric Multiplex FISH (TM-FISH) Strategies
Henegariu, O; Artan, S; Greally, J M; Chen, X-N; Korenberg, J R; Vance, G H; Stubbs, L; Bray-Ward, P; Ward, D C
Experimental data published in recent years showed that up to 10% of all cases with mild to severe idiopathic mental retardation may result from small rearrangements of the subtelomeric regions of human chromosomes. To detect such cryptic translocations, we developed a ''telomeric'' multiplex FISH assay, using a set of previously published and commercially available subtelomeric probes. This set of probes includes 41 cosmid/PAC/P1 clones located from less than 100kb to about 1 Mb from the end of the chromosomes. Similarly, a published mouse probe set, comprised of BACs hybridizing to the closest known marker toward the centromere and telomere of each mouse chromosome, was used to develop a mouse-specific ''telomeric'' M-FISH. Three different combinatorial labeling strategies were used to simultaneously detect all human sub-telomeric regions on one slide. The simplest approach uses only three fluors, and can be performed in laboratories lacking sophisticated imaging equipment or personnel highly trained in cytogenetics. A standard fluorescence microscope equipped with only three filters is sufficient. Fluor-dUTPs and labeled probes can be custom-made, thus dramatically reducing costs. Images can be prepared using generic imaging software (Adobe Photoshop), and analysis performed by simple visual inspection.
Rat Genome Database: a unique resource for rat, human, and mouse quantitative trait locus data
Laulederkind, Stanley J. F.; Hayman, G. Thomas; Smith, Jennifer R.; Wang, Shur-Jen; Lowry, Timothy F.; Petri, Victoria; Pons, Jeff De; Tutaj, Marek; Liu, Weisong; Jayaraman, Pushkala; Munzenmaier, Diane H.; Worthey, Elizabeth A.; Dwinell, Melinda R.; Shimoyama, Mary; Jacob, Howard J.
The rat has been widely used as a disease model in a laboratory setting, resulting in an abundance of genetic and phenotype data from a wide variety of studies. These data can be found at the Rat Genome Database (RGD, http://rgd.mcw.edu/), which provides a platform for researchers interested in linking genomic variations to phenotypes. Quantitative trait loci (QTLs) form one of the earliest and core datasets, allowing researchers to identify loci harboring genes associated with disease. These QTLs are not only important for those using the rat to identify genes and regions associated with disease, but also for cross-organism analyses of syntenic regions on the mouse and the human genomes to identify potential regions for study in these organisms. Currently, RGD has data on >1,900 rat QTLs that include details about the methods and animals used to determine the respective QTL along with the genomic positions and markers that define the region. RGD also curates human QTLs (>1,900) and houses >4,000 mouse QTLs (imported from Mouse Genome Informatics). Multiple ontologies are used to standardize traits, phenotypes, diseases, and experimental methods to facilitate queries, analyses, and cross-organism comparisons. QTLs are visualized in tools such as GBrowse and GViewer, with additional tools for analysis of gene sets within QTL regions. The QTL data at RGD provide valuable information for the study of mapped phenotypes and identification of candidate genes for disease associations. PMID:23881287
Immunostaining of oxidized DJ-1 in human and mouse brains.
Saito, Yoshiro; Miyasaka, Tomohiro; Hatsuta, Hiroyuki; Takahashi-Niki, Kazuko; Hayashi, Kojiro; Mita, Yuichiro; Kusano-Arai, Osamu; Iwanari, Hiroko; Ariga, Hiroyoshi; Hamakubo, Takao; Yoshida, Yasukazu; Niki, Etsuo; Murayama, Shigeo; Ihara, Yasuo; Noguchi, Noriko
DJ-1, the product of a causative gene of a familial form of Parkinson disease, undergoes preferential oxidation of Cys106 (cysteine residue at position 106) under oxidative stress. Using specific monoclonal antibodies against Cys106 oxidized DJ-1 (oxDJ-1), we examined oxDJ-1 immunoreactivity in brain sections from DJ-1 knockout and wild-type mice and in human brain sections from cases classified into different Lewy body stages of Parkinson disease and Parkinson disease with dementia. Oxidized DJ-1 immunoreactivity was prominently observed in neuromelanin-containing neurons and neuron processes of the substantia nigra; Lewy bodies also showed oxDJ-1 immunoreactivity. Oxidized DJ-1 was also detected in astrocytes in the striatum, in neurons and glia in the red nucleus, and in the inferior olivary nucleus, all of which are related to regulation of movement. These observations suggest the relevance of DJ-1 oxidation to homeostasis in multiple brain regions, including neuromelanin-containing neurons of the substantia nigra, and raise the possibility that oxDJ-1 levels might change during the progression of Lewy body-associated neurodegenerative diseases. PMID:24918637
Rabies virus infects mouse and human lymphocytes and induces apoptosis.
Thoulouze, M I; Lafage, M; Montano-Hirose, J A; Lafon, M
Attenuated and highly neurovirulent rabies virus strains have distinct cellular tropisms. Highly neurovirulent strains such as the challenge virus standard (CVS) are highly neurotropic, whereas the attenuated strain ERA also infects nonneuronal cells. We report that both rabies virus strains infect activated murine lymphocytes and the human lymphoblastoid Jurkat T-cell line in vitro. The lymphocytes are more permissive to the attenuated ERA rabies virus strain than to the CVS strain in both cases. We also report that in contrast to that of the CVS strain, ERA viral replication induces apoptosis of infected Jurkat T cells, and cell death is concomitant with viral glycoprotein expression, suggesting that this protein has a role in the induction of apoptosis. Our data indicate that (i) rabies virus infects lymphocytes, (ii) lymphocyte infection with the attenuated rabies virus strain causes apoptosis, and (iii) apoptosis does not hinder rabies virus production. In contrast to CVS, ERA rabies virus and other attenuated rabies virus vaccines stimulate a strong immune response and are efficient live vaccines. The paradoxical finding that a rabies virus triggers a strong immune response despite the fact that it infects lymphocytes and induces apoptosis is discussed in terms of the function of apoptosis in the immune response. PMID:9311815
Human XIST yeast artificial chromosome transgenes show partial X inactivation center function in mouse embryonic stem cells.
Heard, E; Mongelard, F; Arnaud, D; Chureau, C; Vourc'h, C; Avner, P
Initiation of X chromosome inactivation requires the presence, in cis, of the X inactivation center (XIC). The Xist gene, which lies within the XIC region in both human and mouse and has the unique property of being expressed only from the inactive X chromosome in female somatic cells, is known to be essential for X inactivation based on targeted deletions in the mouse. Although our understanding of the developmental regulation and function of the mouse Xist gene has progressed rapidly, less is known about its human homolog. To address this and to assess the cross-species conservation of X inactivation, a 480-kb yeast artificial chromosome containing the human XIST gene was introduced into mouse embryonic stem (ES) cells. The human XIST transcript was expressed and could coat the mouse autosome from which it was transcribed, indicating that the factors required for cis association are conserved in mouse ES cells. Cis inactivation as a result of human XIST expression was found in only a proportion of differentiated cells, suggesting that the events downstream of XIST RNA coating that culminate in stable inactivation may require species-specific factors. Human XIST RNA appears to coat mouse autosomes in ES cells before in vitro differentiation, in contrast to the behavior of the mouse Xist gene in undifferentiated ES cells, where an unstable transcript and no chromosome coating are found. This may not only reflect important species differences in Xist regulation but also provides evidence that factors implicated in Xist RNA chromosome coating may already be present in undifferentiated ES cells. PMID:10359800
Biophysical Properties of ?C-Crystallin in Human and Mouse Eye Lens: The Role of Molecular Dipoles
Purkiss, Andrew G.; Bateman, Orval A.; Wyatt, Keith; Wilmarth, Phillip A.; David, Larry L.; Wistow, Graeme J.; Slingsby, Christine
Summary The eye lens is packed with soluble crystallin proteins, providing a lifetime of transparency and light refraction. ?-crystallins are major components of the dense, high refractive index central regions of the lens and generally have high solubility, high stability and high levels of cysteines. Human ?C belongs to a group of ?-crystallins with a pair of cysteines at positions 78 and 79. Unlike other ?-crystallins it has relatively low solubility, whereas mouse ?C, which has the exposed C79 replaced with arginine, and a novel mouse splice variant, ?Cins, are both highly soluble. Furthermore, human ?C is extremely stable, while the mouse orthologs are less stable. Evolutionary pressure may have favoured stability over solubility for human ?C and the reverse for the orthologs in the mouse. Mutation of C79 to R79, in human ?C, greatly increased solubility, however neither form produced crystals. Remarkably, when the human ?D R36S crystallization cataract mutation was mimicked in human ?C-crystallin, the solubility of ?C was dramatically increased, although it still did not crystallize. The highly soluble mouse ?C-crystallin did crystallize. Its X-ray structure was solved and used in homology modelling of human ?C, and its mutants C79R and R36S. The human ?D R36S mutant was also modelled from human ?D coordinates. Molecular dynamics simulation of the six molecules in the solution state showed that the human ?Cs differed from ?Ds in domain pairing, behaviour that correlates with interface sequence changes. When the fluctuations of the calculated molecular dipoles, for the six structures, over time were analysed, characteristic patterns for soluble ?C and ?D proteins were observed. Individual sequence changes that increase or decrease solubility correlated well with changes in the magnitude and direction of these dipoles. It is suggested that changes in surface residues have allowed adaptation for the differing needs of human and mouse lenses. PMID:17659303
Carbamoyl phosphate synthetase-1 is a rapid turnover biomarker in mouse and human acute liver injury.
Weerasinghe, Sujith V W; Jang, You-Jin; Fontana, Robert J; Omary, M Bishr
Several serum markers are used to assess hepatocyte damage, but they have limitations related to etiology specificity and prognostication. Identification of novel hepatocyte-specific biomarkers could provide important prognostic information and better pathogenesis classification. We tested the hypothesis that hepatocyte-selective biomarkers are released after subjecting isolated mouse hepatocytes to Fas-ligand-mediated apoptosis. Proteomic analysis of hepatocyte culture medium identified the mitochondrial matrix protein carbamoyl phosphate synthetase-1 (CPS1) among the most readily detected proteins that are released by apoptotic hepatocytes. CPS1 was also detected in mouse serum upon acute challenge with Fas-ligand or acetaminophen and in hepatocytes upon hypoosmotic stress, independent of hepatocyte caspase activation. Furthermore, CPS1 was observed in sera of mice chronically fed the hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Mouse CPS1 detectability was similar in serum and plasma, and its half-life was 126 � 9 min. Immune staining showed that CPS1 localized to mouse hepatocytes but not ductal cells. Analysis of a few serum samples from patients with acute liver failure (ALF) due to acetaminophen, Wilson disease, or ischemia showed readily detectable CPS1 that was not observed in several patients with chronic viral hepatitis or in control donors. Notably, CPS1 rapidly decreased to undetectable levels in sera of patients with acetaminophen-related ALF who ultimately recovered, while alanine aminotransferase levels remained elevated. Therefore, CPS1 becomes readily detectable upon hepatocyte apoptotic and necrotic death in culture or in vivo. Its abundance and short serum half-life, compared with alanine aminotransferase, suggest that it may be a useful prognostic biomarker in human and mouse liver injury. PMID:24924744
Secretion of human protein C in mouse milk.
Park, Chae-Won; Kang, Myung-Hwa; Min, Kwan-Sik
To determine the production of recombinant human protein C (rec-hPC) in milk, we created two homozygous mice lines for the goat ?-casein/hPC transgene. Females and males of both lines (#10 and #11) displayed normal growth, fertility, and lactated normally. The copy number of the transgene was about fivefold higher in #10 line as compared to #11 line. mRNA expression of the transgene was only detected in the mammary glands of both lines. Furthermore, mRNA expression was fourfold higher on day 7 than on day 1 during lactation. Northern blot analysis of mRNA expression in the #10 line of transgenic (Tg) mice indicated a strong expression of the transgene in the mammary glands after seven days of lactation. Comparison of rec-hPC protein level with that of mRNA in the mammary glands showed a very similar pattern. A 52-kDa band corresponding to the hPC protein was strongly detected in mammary glands of the #10 line during lactation. We also detected two bands of heavy chain and one weak band of light chain in the milk of the #10 and #11 lines. One single band at 52 kDa was detected from CHO cells transfected with hPC cDNA. hPC was mainly localized in the alveolar epithelial cell of the mammary glands. The protein is strongly expressed in the cytoplasm of the cultured mammary gland tissue. hPC protein produced in milk ranged from 2 to 28 ng/mL. These experiments indicated that rec-hPC can be produced at high levels in mice mammary glands. PMID:25749471
Epstein-Barr-based episomal chromosomes shuttle 100 kb of self-replicating circular human DNA in mouse cells
Kelleher, Z.T.; Fu, H.; Livanos, E.; Wendelburg, B.; Gulino, S.; Vos, J.M. [Univ. of North Carolina, Chapel Hill, NC (United States)] [Univ. of North Carolina, Chapel Hill, NC (United States)
The authors describe the microcell fusion transfer of 100--200 kb self-replicating circular human minichromosomes from human into mouse cells. This experimental approach is illustrated through the shuttling of the latent 170 kb double-stranded DNA genome from the human herpesvirus, Epstein-Barr virus, into nonpermissive rodent cells. Using this interspecies transfer strategy, circular episomes carrying 95--105 kb of human DNA were successfully established at low copy number in mouse A9 cells. Selected episomes were stably maintained for 6 months, and unselected episomes were characterized by a 95% episomal retention per cell division. The establishment of a mouse artificial episomal chromosome system should facilitate evolutionary and therapeutic studies of large human DNA in rodent genetic backgrounds.
Characterization of the human PAP1 gene and its homologue possible involvement in mouse embryonic development.
Shu, Kun-Xian; Wu, Li-Xiang; Xie, Yong-Fang; Zhao, Jin-Feng; Liang, Yi-Long; Li, Biao
We have identified PAP1 gene, a novel member of the immunoglobulin superfamily (IGSF) from U251-pTet-p53 cell line, which carried a wild-type p53 transgene. The gene has been localised to chromosome 16p12-13. Alignment of the predicted protein sequence for Human, Pan troglodytes, Canis, Mus musculus and Gallus gallus revealed it was highly conserved. Its homologue, IGSF6, possible involves in mouse embryonic development. The presence of IGSF6 specific transcript was detected by Northern blot in the RNAs extracted from 11 to 14 day postconception. IGSF6 expression is different in mouse embryos of the different ages. In situ hybridization performed on mice embryos sections showed the differential presence of IGSF6 in developing lung and kidney. This structure and differential expression suggests a function involvement in embryonic development, perhaps involvement in cell proliferation. PMID:16837177
Mapping TNNC1, the gene that encodes cardiac troponin I in the human and the mouse
Bermingham, N.; Hernandez, D.; Fisher, E.M.C. [St. Mary`s Hospital Medical School, London (United Kingdom)] [and others] [St. Mary`s Hospital Medical School, London (United Kingdom); and others
We have mapped the TNNC1 gene, whose protein product is the cardiac TnI protein. TnI is one of the proteins that makes up the troponin complex, which mediates the response of muscle to calcium ions. The human TNNC1 locus had been assigned to a large region of chromosome 19, and we have refined the mapping position to the distal end of the chromosome by amplification of DNAs from a chromosome 19 mapping panel. We have also mapped the mouse Tnnc1 locus, by following the segregation of an intron sequence through DNAs from the European Interspecific Backcross. Tnnc1 maps close to the centromere on mouse chromosome 7. 18 refs., 3 figs., 1 tab.
Human nerve xenografting in nude mouse: Experimental study of graft revascularization
Duprez, K.; Bour, C.; Merle, M.; Duprez, A. (Hopital Jeanne d'Arc, Dommartin-les-Toul (France))
In the nude mouse, the congenital absence of T lymphocytes makes it possible to implant human nerve grafts without rejection or iatrogenic modifications (by immunosuppression) of human and murine tissues. Medial antebrachial cutaneous nerves were harvested from human cadavers 1-18 hours after death. These nerve grafts were implanted using different techniques in nude mice. All the grafts were macroscopically and microscopically revascularized 3 days after implantation. The modifications in time of this vascularization could be studied with precision through the use of repeated biopsies. The absence of human blood group antigens on the neovessel endothelium suggested a murine origin for angiogenesis. In situ DNA hybridizations with human and mouse DNA confirmed this origin. The topography of the revascularization (maximal in the perineurium and endoneurium) and the almost complete absence of human cells other than Schwann cells in the grafts at the peak of angiogenesis (26 days after grafting) suggested that Schwann cells had a determining role in graft vascularization. The irradiation of the nerve grafts with a dose of 30 grays before implantation did not modify significantly their histologic appearance compared to the control group, whereas an irradiation of 60 grays led to massive lesions. The neurotization of murine axons led to chimerical structures of normal histologic appearance, with vascularization similar to that observed in nonneurotized nerves. Through chimerism (human Schwann cells, murine vessels and axons) this model makes it possible to dissociate the respective role of the host and of the nerve graft in angiogenesis and suggests the existence of growth factors produced by the human Schwann cells.
Complement-mediated antiserum cytotoxic reactions to human chromosome 7 coded antigen(s): immunoselection of rearranged human chromosome 7 in human-mouse somatic cell hybrids
Immunoselection via complement-dependent lysis of human-mouse somatic cell hybrids containing chromosome 7, with antisera reactive to cell surface antigen(s) coded for by chromosome 7, has resulted in growth of somatic cell hybrids containing rearranged human chromosome 7s. Investigation of these hybrids has localized the gene(s) coding for the relevant cell surface antigen(s) to the short arm of human chromosome 7. The simian virus 40 integration site and the gene coding for human beta-glucuronidase appear to be localized to the long arm of chromosome 7 in this hybrid clone. PMID:188970
Humanized Mouse Model of Thrombosis is Predictive of the Clinical Efficacy of Antiplatelet Agents
Magallon, Jorge; Chen, Jianchun; Rabbani, Leroy; Dangas, George; Yang, Jing; Bussel, James; Diacovo, Thomas
Background In vivo testing of novel antiplatelet agents requires informative biomarkers. By genetically modifying mouse von Willebrand Factor (VWFR1326H), we have developed a small animal model that supports human but not mouse platelet-mediated thrombosis. Here we evaluate the use of this biological platform as a pharmacodynamic (PD) biomarker for antithrombotic therapies. Methods and Results The antithrombotic effects of several ?IIb?3 inhibitors were determined in VWFR1326H mutant mice infused with human platelets. Administration of abciximab, eptifibatide, or tirofiban at doses recommended for percutaneous coronary intervention (per kg of body weight) significantly reduced human platelet-mediated thrombus formation in laser-injured arterioles by >75% (P<0.001). By contrast clot size in WT control animals remained essentially unchanged (P>0.05), results consistent with observed species differences in IC50 values obtained by aggregometry. To further demonstrate that our biological platform is unique from standard mouse models, we evaluated the thrombogenic potential of platelets from healthy volunteers before and after clopidogrel therapy. Consistent with the antithrombotic effect of this agent, platelets post-drug administration formed smaller thrombi than cells prior to instituting therapy and were less responsive to ADP-induced aggregation (P<0.001). Conclusions The ability of ?IIb?3 and P2Y12 inhibitors to limit human platelet clot formation at doses recommended by ACC/AHA suggests that VWFR1326H mutant mice can serve as both a PD and functional response biomarker, attributes essential for not only expediting drug development but also for designing clinical studies. PMID:21220740
The mouse rumpshaker mutation of the proteolipid protein in human X-linked recessive spastic paraplegia
Kobayashi, H.; Hoffman, E.P.; Matise, T.C. [and others
X-linked recessive spastic paraplegia is a rare neurodegenerative disorder characterized by slowly progressive weakness and spasticity of the lower extremities. We have recently genetically analyzed the original X-linked recessive spastic paraplegia family reported by Johnston and McKusick in 1962. We employed a fluorescent multiplex CA repeat strategy using a 22 locus, 10 cM framework map of the human X chromosome and localized the gene within a 36 cM region of Xq2l.3-q24 which includes the PLP locus. Saugier-Veber et al. recently reported a point mutation (His139Tyr) in exon 3B of the PLP gene in an X-linked recessive spastic paraplegia family (SPG2). This family shows no optic atrophy, in contrast to the family we have studied. This data showed that SPG2 and Pelizaeus-Merzbacher disease were allelic disorders. We investigated the PLP gene as a candidate gene for the original X-linked recessive spastic paraplegia family using SSCP and direct sequencing methods. We found a point mutation (T to C) in exon 4 of affected males which alters the amino-acid (Ile to Thr) at residue 186. This change was absent in the unaffected males of the family and in 40 unrelated control females (80 X chromosomes). Surprisingly, this mutation is identical to the mutation previously identified in the rumpshaker mouse model. The complete homology between both the mouse and human PLP sequence, and the mouse rumpshaker mutation and human spastic paraplegia mutation in our family, permit direct parallels to be drawn with regards to pathophysiology. Our data indicates that the well-documented and striking clinical differences between Pelizaeus-Merzbacher disease and X-linked recessive spastic paraplegia is due to the specific effect of different mutations of the human PLP gene on oligodendrocyte differentiation and development and on later myelin production and maintenance.
Interference by human anti-mouse antibodies in CA 125 assay after immunoscintigraphy: Anti-idiotypic antibodies not neutralized by mouse IgG but removed by chromatography
Turpeinen, U.; Lehtovirta, P.; Alfthan, H.; Stenman, U.H. (Helsinki Univ. Central Hospital (Finland))
Falsely increased concentrations of the ovarian carcinoma-associated antigen, CA 125, were measured by a monoclonal antibody (MAb)-based double determinant immunoradiometric assay (IRMA) in patients who developed antibodies to mouse immunoglobulins (IgGs) after receiving injections of the same MAb as is used in the CA 125 IRMA. Addition of undiluted mouse serum or purified mouse IgG to the assay mixture failed to eliminate the falsely increased CA 125 concentrations in most of the samples, owing to the presence of anti-idiotype antibody. Because of their anti-idiotypic nature, the human anti-mouse antibodies (HAMAS) had only little effect on other immunometric assays, and this effect could be completely eliminated by addition of mouse IgG. To eliminate the effect of HAMA on the CA 125 assay, we studied the ability of various chromatographic methods to separate the interfering HAMA from CA 125. For measuring HAMA in serum and chromatographic fractions we developed a time-resolved fluoroimmunoassay. Adequate separation of CA 125 and HAMA was achieved by affinity chromatography of patients' sera with solid-phase Protein A, Protein G, cation-exchange chromatography on Mono S, and gel filtration on Superose 6. These results demonstrate that the interference can effectively be removed by rather simple chromatographic procedures.
Modular expression analysis reveals functional conservation between human Langerhans cells and mouse cross-priming dendritic cells.
Artyomov, Maxim N; Munk, Adiel; Gorvel, Laurent; Korenfeld, Daniel; Cella, Marina; Tung, Thomas; Klechevsky, Eynav
Characterization of functionally distinct dendritic cell (DC) subsets in mice has fueled interest in whether analogous counterparts exist in humans. Transcriptional modules of coordinately expressed genes were used for defining shared functions between the species. Comparing modules derived from four human skin DC subsets and modules derived from the Immunological Genome Project database for all mouse DC subsets revealed that human Langerhans cells (LCs) and the mouse XCR1(+)CD8?(+)CD103(+) DCs shared the class I-mediated antigen processing and cross-presentation transcriptional modules that were not seen in mouse LCs. Furthermore, human LCs were enriched in a transcriptional signature specific to the blood cross-presenting CD141/BDCA-3(+) DCs, the proposed equivalent to mouse CD8?(+) DCs. Consistent with our analysis, LCs were highly adept at inducing primary CTL responses. Thus, our study suggests that the function of LCs may not be conserved between mouse and human and supports human LCs as an especially relevant therapeutic target. PMID:25918340
Chromosomal assignment of the genes for proprotein convertases PC4, PC5, and PACE 4 in mouse and human
Mbikay, M.; Seidah, N.G.; Chretien, M. [Univ. of Montreal, Quebec (Canada)] [and others] [Univ. of Montreal, Quebec (Canada); and others
The genes for three subtilisin/kexin-like proprotein convertases, PC4, PC5, and PACE4, were mapped in the mouse by RFLP analysis of a DNA panel from a (C57BL/6JEi x SPRET/Ei) F{sub 1} x SPRET/Ei backcross. The chromosomal locations of the human homologs were determined by Southern blot analysis of a DNA panel from human-rodent somatic cell hybrids, most of which contained a single human chromosome each. The gene for PC4 (Pcsk4 locus) mapped to mouse chromosome 10, close to the Adn (adipsin, a serine protease) locus and near the Amh (anti-Mullerian hormone) locus; in a human, the gene was localized to chromosome 19. The gene for PC5 (Pcsk5 locus) mapped to mouse chromosome 19 close to the Lpc1 (lipoacortin-1) locus and, in human, was localized to chromosome 9. The gene for PACE4 (Pcsk6 locus) mapped to mouse chromosome 7, at a distance of 13 cM from the Pcsk3 locus, which specifies furin, another member of this family of enzymes previoulsy mapped to this chromosome. This is in concordance with the known close proximity of these two loci in the homologous region on human chromosome 15q25-qter. Pcsk3 and Pcsk6 mapped to a region of mouse chromosome 7 that has been associated cytogenetically with postnatal lethality in maternal disomy, suggesting that these genes might be candidates for imprinting. 43 refs., 3 figs., 2 tabs.
Cytotoxic effects of propiconazole and its metabolites in mouse and human hepatoma cells and primary mouse hepatocytes
Abstract: Propiconazole is a triazole-containing fungicide that is used agriculturally on grasses, fruits, grains, seeds, hardwoods, and conifers. Propiconazole is a mouse liver hepatotoxicant and a hepatocarcinogen and has adverse reproductive and developmental toxicities in exp...
A method for mutagenesis of mouse mtDNA and a resource of mouse mtDNA mutations for modeling human pathological conditions.
Fayzulin, Rafik Z; Perez, Michael; Kozhukhar, Natalia; Spadafora, Domenico; Wilson, Glenn L; Alexeyev, Mikhail F
Mutations in human mitochondrial DNA (mtDNA) can cause mitochondrial disease and have been associated with neurodegenerative disorders, cancer, diabetes and aging. Yet our progress toward delineating the precise contributions of mtDNA mutations to these conditions is impeded by the limited availability of faithful transmitochondrial animal models. Here, we report a method for the isolation of mutations in mouse mtDNA and its implementation for the generation of a collection of over 150 cell lines suitable for the production of transmitochondrial mice. This method is based on the limited mutagenesis of mtDNA by proofreading-deficient DNA-polymerase ? followed by segregation of the resulting highly heteroplasmic mtDNA population by means of intracellular cloning. Among generated cell lines, we identify nine which carry mutations affecting the same amino acid or nucleotide positions as in human disease, including a mutation in the ND4 gene responsible for 70% of Leber Hereditary Optic Neuropathies (LHON). Similar to their human counterparts, cybrids carrying the homoplasmic mouse LHON mutation demonstrated reduced respiration, reduced ATP content and elevated production of mitochondrial reactive oxygen species (ROS). The generated resource of mouse mtDNA mutants will be useful both in modeling human mitochondrial disease and in understanding the mechanisms of ROS production mediated by mutations in mtDNA. PMID:25820427
A method for mutagenesis of mouse mtDNA and a resource of mouse mtDNA mutations for modeling human pathological conditions
Fayzulin, Rafik Z.; Perez, Michael; Kozhukhar, Natalia; Spadafora, Domenico; Wilson, Glenn L.; Alexeyev, Mikhail F.
Mutations in human mitochondrial DNA (mtDNA) can cause mitochondrial disease and have been associated with neurodegenerative disorders, cancer, diabetes and aging. Yet our progress toward delineating the precise contributions of mtDNA mutations to these conditions is impeded by the limited availability of faithful transmitochondrial animal models. Here, we report a method for the isolation of mutations in mouse mtDNA and its implementation for the generation of a collection of over 150 cell lines suitable for the production of transmitochondrial mice. This method is based on the limited mutagenesis of mtDNA by proofreading-deficient DNA-polymerase ? followed by segregation of the resulting highly heteroplasmic mtDNA population by means of intracellular cloning. Among generated cell lines, we identify nine which carry mutations affecting the same amino acid or nucleotide positions as in human disease, including a mutation in the ND4 gene responsible for 70% of Leber Hereditary Optic Neuropathies (LHON). Similar to their human counterparts, cybrids carrying the homoplasmic mouse LHON mutation demonstrated reduced respiration, reduced ATP content and elevated production of mitochondrial reactive oxygen species (ROS). The generated resource of mouse mtDNA mutants will be useful both in modeling human mitochondrial disease and in understanding the mechanisms of ROS production mediated by mutations in mtDNA. PMID:25820427
Mouse and human blastocyst-derived stem cells: vive les differences.
Rossant, Janet
Lessons learned from conserved vertebrate developmental pathways have catalyzed rapid advances in pluripotent stem cell differentiation towards therapeutically relevant cell types. The most highly conserved phases of development are associated with the early patterning of the body plan - the so-called phylotypic stage. Both prior to and after this stage there is much more divergence across species. Developmental differences between human and mouse at the blastocyst and early post-implantation stages might help explain the differences among the different stem cell lines derived from these embryos. A better understanding of these early stages of human development will aid our ability to generate and manipulate human stem cells and their derivatives. PMID:25516964
Defined conditions for the isolation and expansion of basal prostate progenitor cells of mouse and human origin.
H�fner, Thomas; Eisen, Christian; Klein, Corinna; Rigo-Watermeier, Teresa; Goeppinger, Stephan M; Jauch, Anna; Schoell, Brigitte; Vogel, Vanessa; Noll, Elisa; Weichert, Wilko; Baccelli, Ir�ne; Schillert, Anja; Wagner, Steve; Pahernik, Sascha; Sprick, Martin R; Trumpp, Andreas
Methods to isolate and culture primary prostate epithelial stem/progenitor cells (PESCs) have proven difficult and ineffective. Here, we present a method to grow and expand both murine and human basal PESCs long term in serum- and feeder-free conditions. The method enriches for adherent mouse basal PESCs with a Lin(-)SCA-1(+)CD49f(+)TROP2(high) phenotype. Progesterone and sodium selenite are additionally required for the growth of human Lin(-)CD49f(+)TROP2(high) PESCs. The gene-expression profiles of expanded basal PESCs show similarities to ESCs, and NF-kB function is critical for epithelial differentiation of sphere-cultured PESCs. When transplanted in combination with urogenital sinus mesenchyme, expanded mouse and human PESCs generate ectopic prostatic tubules, demonstrating their stem cell activity in vivo. This novel method will facilitate the molecular, genomic, and functional characterization of normal and pathologic prostate glands of mouse and human origin. PMID:25702639
Validation of a multiplex electrochemiluminescent immunoassay platform in human and mouse samples
Bastarache, J.A.; Koyama, T.; Wickersham, N.E; Ware, L.B.
Despite the widespread use of multiplex immunoassays, there are very few scientific reports that test the accuracy and reliability of a platform prior to publication of experimental data. Our laboratory has previously demonstrated the need for new assay platform validation prior to use of biologic samples from large studies in order to optimize sample handling and assay performance. In this study, our goal was to test the accuracy and reproducibility of an electrochemiluminescent multiplex immunoassay platform (Meso Scale Discovery, MSD�) and compare this platform to validated, singleplex immunoassays (R&D Systems�) using actual study subject (human plasma and mouse bronchoalveolar lavage fluid (BALF) and plasma) samples. We found that the MSD platform performed well on intra- and inter-assay comparisons, spike and recovery and cross-platform comparisons. The mean intra-assay CV% and range for MSD was 3.49 (0.0-10.4) for IL-6 and 2.04 (0.1-7.9) for IL-8. The correlation between values for identical samples measured on both MSD and R&D was R=0.97 for both analytes. The mouse MSD assay had a broader range of CV% with means ranging from 9.5-28.5 depending on the analyte. The range of mean CV% was similar for single plex ELISAs at 4.3-23.7 depending on the analyte. Regardless of species or sample type, CV% was more variable at lower protein concentrations. In conclusion, we validated a multiplex electrochemiluminscent assay system and found that it has superior test characteristics in human plasma compared to mouse BALF and plasma. Both human and MSD assays compared favorably to well-validated singleplex ELISA's PMID:24768796
Biochemical and Morphological Consequences of Human ?-Synuclein Expression in a Mouse ?-Synuclein Null Background
Prasad, Kavita; Tarasewicz, Elizabeth; Strickland, Pamela A. Ohman; O�Neill, Michael; Mitchell, Stephen N.; Merchant, Kalpana; Tep, Samnang; Hilton, Kathryn; Datwani, Akash; Buttini, Manuel; Mueller-Steiner, Sarah; Richfield, Eric K.
A consensus about the functions of human wild-type or mutated ?-synuclein (?SYN) is lacking. Both forms of ?SYN are implicated in Parkinson�s disease, whereas the wild-type form is implicated in substance abuse. Interactions with other cellular proteins and organelles may meditate its functions. We developed a series of congenic mouse lines containing various allele doses or combinations of the human wild type ?SYN (hw?SYN) or a doubly mutated (A30P*A53T) ?SYN (hm2?SYN) in a C57Bl/6J line spontaneously deleted in mouse ?SYN (C57BL/6JOla). Both transgenes had a functional role in the nigrostriatal system, demonstrated by significant elevations in striatal catecholamines, metabolites, and the enzyme tyrosine hydroxylase compared to null-mice without a transgene. Consequences occurred when the transgenes were expressed at a fraction of the endogenous level. Hemizygous congenic mice did not exhibit any change in the number or size of dopaminergic neurons in the ventral midbrain at nine months of age. Human ?SYN was predominantly located in neuronal cell bodies, neurites, synapses, and in intraneuronal/intraneuritic aggregates. The hm2?SYN transgene resulted in more aggregates and dystrophic neurites than did the hw5 transgene. The hw?SYN transgene resulted in higher expression of two striatal proteins, synaptogamin 7 and UCHL1, compared to the levels of the hm2?SYN transgene. These observations suggest that mutations in ?SYN may impair specific functional domains, leaving others intact. These lines may also be useful for exploring interactions between h?SYN and environmental or genetic risk factors in dopamine-related disorders using a mouse model. PMID:21272100
Transcriptional recapitulation and subversion of embryonic colon development by mouse colon tumor models and human colon cancer
Sergio Kaiser; Young-Kyu Park; Jeffrey L Franklin; Richard B Halberg; Ming Yu; Walter J Jessen; Johannes Freudenberg; Xiaodi Chen; Kevin Haigis; Anil G Jegga; Sue Kong; Bhuvaneswari Sakthivel; Huan Xu; Timothy Reichling; Mohammad Azhar; Gregory P Boivin; Reade B Roberts; Anika C Bissahoyo; Fausto Gonzales; Greg C Bloom; Steven Eschrich; Scott L Carter; Jeremy E Aronow; John Kleimeyer; Michael Kleimeyer; Vivek Ramaswamy; Stephen H Settle; Braden Boone; Shawn Levy; Jonathan M Graff; Thomas Doetschman; Joanna Groden; William F Dove; David W Threadgill; Timothy J Yeatman; Robert J Coffey Jr; Bruce J Aronow
BACKGROUND: The expression of carcino-embryonic antigen by colorectal cancer is an example of oncogenic activation of embryonic gene expression. Hypothesizing that oncogenesis-recapitulating-ontogenesis may represent a broad programmatic commitment, we compared gene expression patterns of human colorectal cancers (CRCs) and mouse colon tumor models to those of mouse colon development embryonic days 13.5-18.5. RESULTS: We report here that 39 colon tumors
Features and Trend of Loss of Promoter-Associated CpG Islands in the Human and Mouse Genomes
Cizhong Jiang; Leng Han; Bing Su; Wen-Hsiung Li; Zhongming Zhao
CpG islands (CGIs) are often considered as gene markers, but the number of CGIs varies among mammalian genomes that have similar numbers of genes. In this study, we investigated the distribution of CGIs in the promoter regions of 3,197 human-mouse orthologous gene pairs and found that the mouse genome has notably fewer CGIs in the promoter regions and less pronounced
Hairless Mouse Skin is Limited as a Model for Assessing the Effects of Penetration Enhancers in Human Skin
John Russell Bond; Brian William Barry
The permeability coefficient of 5-fluorouracil through human abdominal and hairless mouse skins was used as an indicator of the relative effects of 12-h pretreatment of the skins with either penetration-enhancer mixtures [including laurocapram (Azone), decylmethylsulfoxide, oleic acid, and propylene glycol] or saline (control). After treatment with saline, fluxes of 5-fluorouracil through the two skin types were similar, but the mouse
Preclinical evaluation of human secretoglobin 3A2 in mouse models of lung development and fibrosis
Cai, Yan; Winn, Melissa E.; Zehmer, John K.; Gillette, William K.; Lubkowski, Jacek T.; Pilon, Aprile L.
Secretoglobin (SCGB) 3A2 is a member of the SCGB gene superfamily of small secreted proteins, predominantly expressed in lung airways. We hypothesize that human SCGB3A2 may exhibit anti-inflammatory, growth factor, and antifibrotic activities and be of clinical utility. Recombinant human SCGB3A2 was expressed, purified, and biochemically characterized as a first step to its development as a therapeutic agent in clinical settings. Human SCGB3A2, as well as mouse SCGB3A2, readily formed a dimer in solution and exhibited novel phospholipase A2 inhibitory activity. This is the first demonstration of any quantitative biochemical measurement for the evaluation of SCGB3A2 protein. In the mouse as an experimental animal, human SCGB3A2 exhibited growth factor activity by promoting embryonic lung development in both ex vivo and in vivo systems and antifibrotic activity in the bleomycin-induced lung fibrosis model. The results suggested that human SCGB3A2 can function as a growth factor and an antifibrotic agent in humans. When SCGB3A2 was administered to pregnant female mice through the tail vein, the protein was detected in the dam's serum and lung, as well as the placenta, amniotic fluids, and embryonic lungs at 10 min postadministration, suggesting that SCGB3A2 readily crosses the placenta. The results warrant further development of recombinant SCGB3A2 as a therapeutic agent in treating patients suffering from lung diseases or preterm infants with respiratory distress. PMID:24213919
Porcine induced pluripotent stem cells may bridge the gap between mouse and human iPS.
Esteban, Miguel A; Peng, Meixiu; Deli, Zhang; Cai, Jie; Yang, Jiayin; Xu, Jianyong; Lai, Liangxue; Pei, Duanqing
Recently, three independent laboratories reported the generation of induced pluripotent stem cells (iPSCs) from pig (Sus scrofa). This finding sums to the growing list of species (mouse, human, monkey, and rat, in this order) for which successful reprogramming using exogenous factors has been achieved, and multiple others are possibly forthcoming. But apart from demonstrating the universality of the network identified by Shinya Yamanaka, what makes the porcine model so special? On one side, pigs are an agricultural commodity and have an easy and affordable maintenance compared with nonhuman primates that normally need to be imported. On the other side, resemblance (for example, size of organs) of porcine and human physiology is striking and because pigs are a regular source of food the ethical concerns that still remain in monkeys are not applicable. Besides, the prolonged lifespan of pigs compared with other domestic species can allow exhaustive follow up of side effects after transplantation. Porcine iPSCs may thus fill the gap between the mouse model, which due to its ease is preferred for mechanistic studies, and the first clinical trials using iPSCs in humans. However, although these studies are relevant and have created significant interest they face analogous problems that we discuss herein together with potential new directions. PMID:20101630
Localization of a human homolog of the mouse pericentrin gene (PCNT) to chromosome 21qter
Chen, Haiming [Univ. of Geneva Medical School (Switzerland)] [Univ. of Geneva Medical School (Switzerland); Gos, A.; Morris, M.A. [Cantonal Hospital, Geneva (Switzerland)] [and others] [Cantonal Hospital, Geneva (Switzerland); and others
Exon trapping was used to identify portions of genes from cosmid DNA of a human chromosome 21-specific library LL21NC02-Q. More than 650 potential exons have been cloned and characterized to date. Among these, 3 trapped {open_quotes}exons{close_quotes} showed strong homology to different regions of the cDNA for the mouse pericentrin (Pcnt) gene, indicating that these 3 exons are portions of a human homolog of the mouse pericentrin gene. With PCR amplification, Southern blot analysis, and FISH, we have mapped this presumed human pericentrin gene (PCNT) to the long arm of chromosome 21 between marker PFKL and 21qter. Pericentrin is a conserved protein component of the filamentous matrix of the centrosome involved in the initial establishment of the organized microtubule array. No candidate hereditary disorder for pericentrin deficiency/abnormality has yet been mapped in the most distal region of 21q; in addition the role of triplication of the pericentrin gene in the pathophysiology or etiology of trisomy 21 is currently unknown. 16 refs., 3 figs.
Expression, purification and characterization of a mouse-human chimeric antibody and chimeric Fab' fragment.
King, D J; Adair, J R; Angal, S; Low, D C; Proudfoot, K A; Lloyd, J C; Bodmer, M W; Yarranton, G T
B72.3 is a mouse monoclonal antibody against a tumour-associated antigen, TAG72, which recognizes breast, ovarian and colorectal tumour tissue. A mouse-human chimeric version of B72.3 has been expressed in Chinese-hamster ovary cells. This molecule has the binding specificity of B72.3 and constant regions from human IgG4. The chimeric B72.3 assembles to intact IgG and recognizes TAG72 as well as B72.3 in competitive binding assays. A proportion of the chimeric B72.3 (approx. 10%) does not form inter-heavy-chain disulphide bonds but still assembles into the IgG tetramer. This appears to be a general property of human IgG4 molecules. Co-expression of the chimeric light chain with a chimeric Fd' gene resulted in the expression of functional Fab'. Very little F(ab')2 is produced, although the Fab' can be oxidized to the dimeric F(ab')2 in vitro. The production of Fab' and F(ab')2 by this method is an attractive alternative to proteolytic digestion of IgG. The ability to produce these molecules in large quantities will allow the production and testing of a range of anti-tumour antibody and antibody fragment conjugates. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. Fig. 6. Fig. 9. Fig. 10. PMID:1736881
A genetic assay is described in which a mouse/human hybrid cell line R3-5 containing a single human chromosome (a monochromosomal hybrid) is used to detect chemically induced aneuploidy. The hybrid cells are deficient in hypoxanthine guanine phosphoribosyltransferase (HGPRT) and ...
A detailed analysis of the erythropoietic control system in the human, squirrel, monkey, rat and mouse
NASA Technical Reports Server (NTRS)
Nordheim, A. W.
The erythropoiesis modeling performed in support of the Body Fluid and Blood Volume Regulation tasks is described. The mathematical formulation of the species independent model, the solutions to the steady state and dynamic versions of the model, and the individual species specific models for the human, squirrel monkey, rat and mouse are outlined. A detailed sensitivity analysis of the species independent model response to parameter changes and how those responses change from species to species is presented. The species to species response to a series of simulated stresses directly related to blood volume regulation during space flight is analyzed.
Efficacy of phthalocyanine tetrasulfonate against mouse-adapted human prion strains.
Abdel-Haq, Hanin; Lu, Mei; Cardone, Franco; Liu, Quan Guo; Puopolo, Maria; Pocchiari, Maurizio
In vitro and in vivo studies have shown that phthalocyanine tetrasulfonate (PcTS), a cyclic tetrapyrrole compound, is an efficient antiscrapie drug. To investigate the spectrum of PcTS against prion diseases, we tested the effect of PcTS on two mouse-adapted human strains. We also tested PcTS in rodents infected with two scrapie strains (139A and 263K). PcTS treatment significantly prolonged mean survival times of all infected animals. These results show that PcTS is effective on different prion strains, confirming its potential use for prion therapy. PMID:19458902
Treatment of NASH with human mesenchymal stem cells in the immunodeficient mouse.
Winkler, Sandra; Christ, Bruno
Nonalcoholic steatohepatitis (NASH) as a severe form of nonalcoholic liver diseases (NAFLD) is one of the prominent liver diseases worldwide. Under favoring conditions it may progress into liver cirrhosis and hepatocellular carcinoma (HCC), which in its end stage strongly requires organ transplantation. Due to the shortage of donor organs, alternative therapeutic approaches like cell therapy treatment are necessary. In this article, an auspicious method of cell therapy with hepatocytic differentiated human mesenchymal stem cells to treat NASH in an immunodeficient mouse model is presented. PMID:25173373
Generation and Characterization of a Transgenic Mouse Carrying a Functional Human ?-Globin Gene with the IVSI-6 Thalassemia Mutation
Mancini, Irene; Lampronti, Ilaria; Salvatori, Francesca; Fabbri, Enrica; Zuccato, Cristina; Cosenza, Lucia C.; Montagner, Giulia; Borgatti, Monica; Altruda, Fiorella; Fagoonee, Sharmila; Carandina, Gianni; Aiello, Vincenzo; Breda, Laura; Rivella, Stefano; Gambari, Roberto
Mouse models that carry mutations causing thalassemia represent a suitable tool to test in vivo new mutation-specific therapeutic approaches. Transgenic mice carrying the ?-globin IVSI-6 mutation (the most frequent in Middle-Eastern regions and recurrent in Italy and Greece) are, at present, not available. We report the production and characterization of a transgenic mouse line (TG-?-IVSI-6) carrying the IVSI-6 thalassemia point mutation within the human ?-globin gene. In the TG-?-IVSI-6 mouse (a) the transgenic integration region is located in mouse chromosome 7; (b) the expression of the transgene is tissue specific; (c) as expected, normally spliced human ?-globin mRNA is produced, giving rise to ?-globin production and formation of a human-mouse tetrameric chimeric hemoglobin mu?-globin2/hu?-globin2 and, more importantly, (d) the aberrant ?-globin-IVSI-6 RNAs are present in blood cells. The TG-?-IVSI-6 mouse reproduces the molecular features of IVSI-6 ?-thalassemia and might be used as an in vivo model to characterize the effects of antisense oligodeoxynucleotides targeting the cryptic sites responsible for the generation of aberrantly spliced ?-globin RNA sequences, caused by the IVSI-6 mutation. These experiments are expected to be crucial for the development of a personalized therapy for ?-thalassemia.
Generation and Characterization of a Transgenic Mouse Carrying a Functional Human ? -Globin Gene with the IVSI-6 Thalassemia Mutation.
Breveglieri, Giulia; Mancini, Irene; Bianchi, Nicoletta; Lampronti, Ilaria; Salvatori, Francesca; Fabbri, Enrica; Zuccato, Cristina; Cosenza, Lucia C; Montagner, Giulia; Borgatti, Monica; Altruda, Fiorella; Fagoonee, Sharmila; Carandina, Gianni; Rubini, Michele; Aiello, Vincenzo; Breda, Laura; Rivella, Stefano; Gambari, Roberto; Finotti, Alessia
Mouse models that carry mutations causing thalassemia represent a suitable tool to test in vivo new mutation-specific therapeutic approaches. Transgenic mice carrying the ?-globin IVSI-6 mutation (the most frequent in Middle-Eastern regions and recurrent in Italy and Greece) are, at present, not available. We report the production and characterization of a transgenic mouse line (TG-?-IVSI-6) carrying the IVSI-6 thalassemia point mutation within the human ?-globin gene. In the TG-?-IVSI-6 mouse (a) the transgenic integration region is located in mouse chromosome 7; (b) the expression of the transgene is tissue specific; (c) as expected, normally spliced human ?-globin mRNA is produced, giving rise to ?-globin production and formation of a human-mouse tetrameric chimeric hemoglobin (mu) ?-globin2/(hu) ?-globin2 and, more importantly, (d) the aberrant ?-globin-IVSI-6 RNAs are present in blood cells. The TG-?-IVSI-6 mouse reproduces the molecular features of IVSI-6 ?-thalassemia and might be used as an in vivo model to characterize the effects of antisense oligodeoxynucleotides targeting the cryptic sites responsible for the generation of aberrantly spliced ?-globin RNA sequences, caused by the IVSI-6 mutation. These experiments are expected to be crucial for the development of a personalized therapy for ?-thalassemia. PMID:26097845
Signal-regulatory protein ? from the NOD mouse binds human CD47 with an exceptionally high affinity���implications for engraftment of human cells
Kwong, Lai Shan; Brown, Marion H; Barclay, A Neil; Hatherley, Deborah
One common way to study human leucocytes and cancer cells in an experimental in vivo situation is to use mice that have been genetically engineered to lack an immune system and prevent human cell rejection. These mice lack CD132 and either RAG2 or the catalytic subunit of the DNA-dependent protein kinase, to make the mice deficient in lymphocytes and natural killer cells. The NOD mouse strain provides a better background for engraftment than other strains due to stronger engagement of the signal-regulatory protein-? (SIRP?) inhibitory receptor with human CD47 (hCD47) resulting in a �don't-eat-me� signal. To determine the molecular parameters that determine this major functional effect in the NOD mouse we measured the affinity of hCD47 for SIRP? from various mouse strains. Human CD47 bound SIRP? from the NOD mouse with an affinity 65 times greater than SIRP? from other mouse strains. This is due mainly to the NOD SIRP? lacking two amino acids in domain 1 compared with other mouse strains. Remarkably the SIRP?(NOD) binds hCD47 with 10 times the affinity of the syngeneic hCD47/hSIRP? interaction. This affinity is outside the normal range for affinities for leucocyte surface protein interactions and raises questions as to what is the optimal affinity of this interaction for engraftment and what other xenogeneic interactions involved in homeostasis may also not be optimal. PMID:24786312
Translation of BAFF Inhibition from Mouse to Non-human Primate and Human
Lachy McLean; Dhaya Seshasayee; Susan L. Kalled; Flavius Martin
\\u000a The identification of BAFF as a fundamental B cell survival factor in mouse and man, its over-expression in certain autoimmune\\u000a disease patient populations, and the discovery of three cognate receptors, stimulated interest in understanding the role of\\u000a BAFF in the pathogenesis of autoimmunity, and designing novel therapeutics to blunt B cell participation in disease pathogenesis\\u000a via blockade of this pathway.
Adaptation of hepatitis C virus to mouse CD81 permits infection of mouse cells in the absence of human entry factors.
Bitzegeio, Julia; Bankwitz, Dorothea; Hueging, Kathrin; Haid, Sibylle; Brohm, Christiane; Zeisel, Mirjam B; Herrmann, Eva; Iken, Marcus; Ott, Michael; Baumert, Thomas F; Pietschmann, Thomas
Hepatitis C virus (HCV) naturally infects only humans and chimpanzees. The determinants responsible for this narrow species tropism are not well defined. Virus cell entry involves human scavenger receptor class B type I (SR-BI), CD81, claudin-1 and occludin. Among these, at least CD81 and occludin are utilized in a highly species-specific fashion, thus contributing to the narrow host range of HCV. We adapted HCV to mouse CD81 and identified three envelope glycoprotein mutations which together enhance infection of cells with mouse or other rodent receptors approximately 100-fold. These mutations enhanced interaction with human CD81 and increased exposure of the binding site for CD81 on the surface of virus particles. These changes were accompanied by augmented susceptibility of adapted HCV to neutralization by E2-specific antibodies indicative of major conformational changes of virus-resident E1/E2-complexes. Neutralization with CD81, SR-BI- and claudin-1-specific antibodies and knock down of occludin expression by siRNAs indicate that the adapted virus remains dependent on these host factors but apparently utilizes CD81, SR-BI and occludin with increased efficiency. Importantly, adapted E1/E2 complexes mediate HCV cell entry into mouse cells in the absence of human entry factors. These results further our knowledge of HCV receptor interactions and indicate that three glycoprotein mutations are sufficient to overcome the species-specific restriction of HCV cell entry into mouse cells. Moreover, these findings should contribute to the development of an immunocompetent small animal model fully permissive to HCV. PMID:20617177
Analysis of glycoprotein E-selectin ligands on human and mouse marrow cells enriched for hematopoietic stem/progenitor cells
Merzaban, Jasmeen S.; Burdick, Monica M.; Gadhoum, S. Zeineb; Dagia, Nilesh M.; Chu, Julia T.; Fuhlbrigge, Robert C.
Although well recognized that expression of E-selectin on marrow microvessels mediates osteotropism of hematopoietic stem/progenitor cells (HSPCs), our knowledge regarding the cognate E-selectin ligand(s) on HSPCs is incomplete. Flow cytometry using E-selectin-Ig chimera (E-Ig) shows that human marrow cells enriched for HSPCs (CD34+ cells) display greater E-selectin binding than those obtained from mouse (lin?/Sca-1+/c-kit+ [LSK] cells). To define the relevant glycoprotein E-selectin ligands, lysates from human CD34+ and KG1a cells and from mouse LSK cells were immunoprecipitated using E-Ig and resolved by Western blot using E-Ig. In both human and mouse cells, E-selectin ligand reactivity was observed at ? 120- to 130-kDa region, which contained two E-selectin ligands, the P-selectin glycoprotein ligand-1 glycoform �CLA,� and CD43. Human, but not mouse, cells displayed a prominent ? 100-kDa band, exclusively comprising the CD44 glycoform �HCELL.� E-Ig reactivity was most prominent on CLA in mouse cells and on HCELL in human cells. To further assess HCELL's contribution to E-selectin adherence, complementary studies were performed to silence (via CD44 siRNA) or enforce its expression (via exoglycosylation). Under physiologic shear conditions, CD44/HCELL-silenced human cells showed striking decreases (> 50%) in E-selectin binding. Conversely, enforced HCELL expression of LSK cells profoundly increased E-selectin adherence, yielding > 3-fold more marrow homing in vivo. These data define the key glycoprotein E-selectin ligands of human and mouse HSPCs, unveiling critical species-intrinsic differences in both the identity and activity of these structures. PMID:21659548
Differential expression and regulation by activin of the neurotrophins BDNF and NT4 during human and mouse ovarian development.
Childs, Andrew J; Bayne, Rosemary A L; Murray, Alison A; Martins Da Silva, Sarah J; Collins, Craig S; Spears, Norah; Anderson, Richard A
The tropomyosin-related kinase (Trk) B neurotrophin receptor is essential for ovarian germ cell survival and primordial follicle formation, but the contributions of its ligands, brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4), are unknown. We have investigated their expression and regulation in developing human and mouse ovaries. BDNF expression increased with increasing gestation, expression of human NTF4 and of both Ntf5 and Bdnf in the mouse was unchanged. Bdnf expression was dramatically lower than Ntf5 in the mouse, but levels were comparable in the human. Human fetal ovarian somatic cells expressed BDNF. Activin A selectively regulated BDNF and Ntf5 expression in human and mouse, respectively, identifying an oocyte/somatic signaling pathway which might mediate the pro-survival effects of activin. These data reveal that expression and regulation of the TrkB ligands are differentially controlled in the developing ovaries of humans and mice, and identify BDNF as a potential regulator of germ cell fate in the human fetal ovary. PMID:20175187
Expression of the human Dp 71 (apo-dystrophin 1) gene from a 760 kb reconstructed human distal DMD YAC transferred to mouse cells
Ommen, G.J.B. van; Heikoop, J.C.; Hogervorst, F.B.L. [Leiden Univ. (Netherlands)] [and others
In a program to re-introduce and study the 2.5 mb human DMD gene in a mouse background, we have first reconstructed the gene on a single YAC by homologous recombination. We are now testing pilot gene transfer of a 760 kb YAC generated during the process and covering the 3{prime} region of the gene. This YAC contains exons 52-79 and thus includes the internal genes of Dp 71 (apo-dystrophin 1) and Dp 116 (apo-dystrophin 2). To facilitate selection in mammalian cells, the YAC was modified by recombinational insertion (retrofitting) of a neomycin-resistance gene in the right vector-arm. This YAC, yneo(18-25)C, was introduced in mouse LA-9 cells by PEG-mediated cell fusion. G418 resistant transformants were characterized by DMD-exon-PCR and Southern blotting. One of the six clones analyzed accommodated the entire intact YAC-DNA. Expression of the human DMD gene was studied by RT-PCR and revealed expression of the human Dp 71 gene but not of the Dp 116 gene in the full-length clone LA-9/3A. Remarkably, differences were observed in the 3{prime} region of the mouse and the human mRNAs, due to alternative splicing of exons 71 (absent in the human mRNA, present in the mouse mRNA) and 78 (present in the human mRNA, absent in the mouse mRNA). The splicing pattern of the human transcript mirrors that of the major product in human blood cells, suggesting that in this murine cell line processing of the human and the mouse DMD transcripts maintains the exon selectivity of the original species.
Novel diet-related mouse model of colon cancer parallels human colon cancer
Prasad, Anil R; Prasad, Shilpa; Nguyen, Huy; Facista, Alexander; Lewis, Cristy; Zaitlin, Beryl; Bernstein, Harris; Bernstein, Carol
AIM: To investigate the close parallels between our novel diet-related mouse model of colon cancer and human colon cancer. METHODS: Twenty-two wild-type female mice (ages 6-8 wk) were fed the standard control diet (AIN-93G) and an additional 22 female mice (ages 6-8 wk) were fed the control diet supplemented with 0.2% deoxycholic acid [diet + deoxycholic acid (DOC)] for 10 mo. Tumors occurred in the colons of mice fed diet + DOC and showed progression to colon cancer [adenocarcinoma (AC)]. This progression is through the stages of tubular adenoma (TA), TA with high grade dysplasia or adenoma with sessile serrated morphology, intramucosal AC, AC stage T1, and AC stage T2. The mouse tumors were compared to human tumors at the same stages by histopathological analysis. Sections of the small and large intestines of mice and humans were evaluated for glandular architecture, cellular and nuclear morphology including cellular orientation, cellular and nuclear atypia, pleomorphism, mitotic activity, frequency of goblet cells, crypt architecture, ulceration, penetration of crypts through the muscularis mucosa and presence of malignant crypts in the muscularis propria. In addition, preserved colonic tissues from genetically similar male mice, obtained from a prior experiment, were analyzed by immunohistochemistry. The male mice had been fed the control diet or diet + DOC. Four molecular markers were evaluated: 8-OH-dG, DNA repair protein ERCC1, autophagy protein beclin-1 and the nuclear location of beta-catenin in the stem cell region of crypts. Also, male mice fed diet + DOC plus 0.007% chlorogenic acid (diet + DOC + CGA) were evaluated for ERCC1, beclin-1 and nuclear location of beta-catenin. RESULTS: Humans with high levels of diet-related DOC in their colons are at a substantially increased risk of developing colon cancer. The mice fed diet + DOC had levels of DOC in their colons comparable to that of humans on a high fat diet. The 22 mice without added DOC in their diet had no colonic tumors while 20 of the 22 mice (91%) fed diet + DOC developed colonic tumors. Furthermore, the tumors in 10 of these mice (45% of mice) included an adenocarcinoma. All mice were free of cancers of the small intestine. Histopathologically, the colonic tumor types in the mice were virtually identical to those in humans. In humans, characteristic aberrant changes in molecular markers can be detected both in field defects surrounding cancers (from which cancers arise) and within cancers. In the colonic tissues of mice fed diet + DOC similar changes in biomarkers appeared to occur. Thus, 8-OH-dG was increased, DNA repair protein ERCC1 was decreased, autophagy protein beclin-1 was increased and, in the stem cell region at the base of crypts there was substantial nuclear localization of beta-catenin as well as increased cytoplasmic beta-catenin. However, in mice fed diet + DOC + CGA (with reduced frequency of cancer) and evaluated for ERCC1, beclin-1, and beta-catenin in the stem cell region of crypts, mouse tissue showed amelioration of the aberrancies, suggesting that chlorogenic acid is protective at the molecular level against colon cancer. This is the first diet-related model of colon cancer that closely parallels human progression to colon cancer, both at the histomorphological level as well as in its molecular profile. CONCLUSION: The diet-related mouse model of colon cancer parallels progression to colon cancer in humans, and should be uniquely useful in model studies of prevention and therapeutics. PMID:25024814
Preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells.
Jozefczuk, Justyna; Drews, Katharina; Adjaye, James
In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)(1) can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial. The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium(2). Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs(3). Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)(4). Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions(5-7). Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established(8). To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells(9), adult skin cells(10), foreskin fibroblasts(11-12), amniotic mesenchymal cells(13)). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A(14). Obviously, there is an evident difference in growth factor production by mouse and human feeder cells. Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf?1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs(15-16). For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs. Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media. PMID:22760161
Development and rescue of human familial hypercholesterolaemia in a xenograft mouse model.
Bissig-Choisat, Beatrice; Wang, Lili; Legras, Xavier; Saha, Pradip K; Chen, Leon; Bell, Peter; Pankowicz, Francis P; Hill, Matthew C; Barzi, Mercedes; Leyton, Claudia Kettlun; Leung, Hon-Chiu Eastwood; Kruse, Robert L; Himes, Ryan W; Goss, John A; Wilson, James M; Chan, Lawrence; Lagor, William R; Bissig, Karl-Dimiter
Diseases of lipid metabolism are a major cause of human morbidity, but no animal model entirely recapitulates human lipoprotein metabolism. Here we develop a xenograft mouse model using hepatocytes from a patient with familial hypercholesterolaemia caused by loss-of-function mutations in the low-density lipoprotein receptor (LDLR). Like familial hypercholesterolaemia patients, our familial hypercholesterolaemia liver chimeric mice develop hypercholesterolaemia and a 'humanized' serum profile, including expression of the emerging drug targets cholesteryl ester transfer protein and apolipoprotein (a), for which no genes exist in mice. We go on to replace the missing LDLR in familial hypercholesterolaemia liver chimeric mice using an adeno-associated virus 9-based gene therapy and restore normal lipoprotein profiles after administration of a single dose. Our study marks the first time a human metabolic disease is induced in an experimental animal model by human hepatocyte transplantation and treated by gene therapy. Such xenograft platforms offer the ability to validate human experimental therapies and may foster their rapid translation into the clinic. PMID:26081744
Distinct glycosyltransferases synthesize E-selectin ligands in human vs. mouse leukocytes
Mondal, Nandini; Buffone Jr., Alexander; Neelamegham, Sriram
The binding of selectins to carbohydrate epitopes expressed on leukocytes is the first step in a multi-step cell adhesion cascade that controls the rate of leukocyte recruitment at sites of inflammation. The glycans that function as selectin-ligands are post-translationally synthesized by the serial action of Golgi resident enzymes called glycosyltransferases (glycoTs). Whereas much of our current knowledge regarding the role of glycoTs in constructing selectin-ligands comes from reconstituted biochemical investigations or murine models, tools to assess the impact of these enzymes on the human ligands are relatively underdeveloped. This is significant since the selectin-ligands, particularly those that bind E-selectin, vary between different leukocyte cell populations and they are also different in humans compared with mice. To address this shortcoming, a recent study by Buffone et al. (2013) outlines a systematic strategy to knockdown upto three glycoTs simultaneously in human leukocytes. The results suggest that the fucosyltransferases (FUTs) regulating selectin-ligand synthesis may be species-specific. In particular, they demonstrate that FUT9 plays a significant role during human, but not mouse, leukocyte-endothelial interactions. Overall, this article discusses the relative roles of the FUTs during human L-, E-, and P-selectin-ligand biosynthesis, and the potential that the knockdown strategy outlined here may assess the role of other glycoTs in human leukocytes also. PMID:23590904
Transplantation of human menstrual blood stem cells to treat premature ovarian failure in mouse model.
Liu, Te; Huang, Yongyi; Zhang, Jian; Qin, Wenxing; Chi, Huiying; Chen, Jiulin; Yu, Zhihua; Chen, Chuan
The incidence of premature ovarian failure (POF), also known as ovarian insufficiency, has been increasing in recent years. Although some treatments are currently available, improved treatment strategies are urgently required. Many researchers have reported that human endometrial stem cells (HuMenSCs), which exhibit stem/progenitor cell properties in vitro repaired damaged cells in vivo. Thus, we aimed to determine whether HuMenSCs can serve as cell therapy tools and be used for the treatment of POF. After treating with cyclophosphamide, on the first estrus period (we predicted mouse estrus cycle was generally 5 days), HuMenSCs were injected into a cyclophosphamide-induced mouse model of POF. The results revealed that the HuMenSCs could survive within POF mouse ovaries for at least 14 days in vivo; further, ovaries of the HuMenSCs-transplanted group expressed higher levels of ovarian markers [AMH, inhibin ?/?, and follicle-stimulating hormone receptor (FSHR)], and the proliferative marker Ki67. In addition, the ovarian weight, plasma E2 level, and the number of normal follicles increased over time in the HuMenSC group compared with the control group. Further, microarray analysis of cDNA expression patterns revealed that, after HuMenSC transplantation, the gene mRNA expression patterns in the ovarian cells following stimulation of the host ovarian niche became increasingly similar to those observed in human ovarian tissue compared with the pretransplantation mRNA expression pattern in HuMenSCs. Hence, we can safely conclude that the mesenchymal stem cell properties and in vivo survival of HuMenSCs make them ideal seed cells for stem cell transplantation in the treatment of POF. PMID:24593672
A humanized mouse model of hereditary 1,25-dihydroxyvitamin D-resistant rickets without alopecia.
Lee, Seong Min; Goellner, Joseph J; O'Brien, Charles A; Pike, J Wesley
The syndrome of hereditary 1,25-dihydroxyvitamin D-resistant rickets (HVDRR) is a genetic disease of altered mineral homeostasis due to mutations in the vitamin D receptor (VDR) gene. It is frequently, but not always, accompanied by the presence of alopecia. Mouse models that recapitulate this syndrome have been prepared through genetic deletion of the Vdr gene and are characterized by the presence of rickets and alopecia. Subsequent studies have revealed that VDR expression in hair follicle keratinocytes protects against alopecia and that this activity is independent of the protein's ability to bind 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. In the present study, we introduced into VDR-null mice a human VDR (hVDR) bacterial artificial chromosome minigene containing a mutation that converts leucine to serine at amino acid 233 in the hVDR protein, which prevents 1,25(OH)2D3 binding. We then assessed whether this transgene recreated features of the HVDRR syndrome without alopecia. RT-PCR and Western blot analysis in one strain showed an appropriate level of mutant hVDR expression in all tissues examined including skin. The hVDR-L233S mutant failed to rescue the aberrant systemic and skeletal phenotype characteristic of the VDR null mouse due to the inability of the mutant receptor to activate transcription after treatment with 1,25(OH)2D3. Importantly, however, neither alopecia nor the dermal cysts characteristic of VDR-null mice were observed in the skin of these hVDR-L233S mutant mice. This study confirms that we have created a humanized mouse model of HVDRR without alopecia that will be useful in defining additional features of this syndrome and in identifying potential novel functions of the unoccupied VDR. PMID:25147982
The VP40 Protein of Marburg Virus Exhibits Impaired Budding and Increased Sensitivity to Human Tetherin following Mouse Adaptation
Feagins, Alicia R.
ABSTRACT The Marburg virus VP40 protein is a viral matrix protein that spontaneously buds from cells. It also functions as an interferon (IFN) signaling antagonist by targeting Janus kinase 1 (JAK1). A previous study demonstrated that the VP40 protein of the Ravn strain of Marburg virus (Ravn virus [RAVV]) failed to block IFN signaling in mouse cells, whereas the mouse-adapted RAVV (maRAVV) VP40 acquired the ability to inhibit IFN responses in mouse cells. The increased IFN antagonist function of maRAVV VP40 mapped to residues 57 and 165, which were mutated during the mouse adaptation process. In the present study, we demonstrate that maRAVV VP40 lost the capacity to efficiently bud from human cell lines, despite the fact that both parental and maRAVV VP40s bud efficiently from mouse cell lines. The impaired budding in human cells corresponds with the appearance of protrusions on the surface of maRAVV VP40-expressing Huh7 cells and with an increased sensitivity of maRAVV VP40 to restriction by human tetherin but not mouse tetherin. However, transfer of the human tetherin cytoplasmic tail to mouse tetherin restored restriction of maRAVV VP40. Residues 57 and 165 were demonstrated to contribute to the failure of maRAVV VP40 to bud from human cells, and residue 57 was demonstrated to alter VP40 oligomerization, as assessed by coprecipitation assay, and to determine sensitivity to human tetherin. This suggests that RAVV VP40 acquired, during adaptation to mice, changes in its oligomerization potential that enhanced IFN antagonist function. However, this new capacity impaired RAVV VP40 budding from human cells. IMPORTANCE Filoviruses, which include Marburg viruses and Ebola viruses, are zoonotic pathogens that cause severe disease in humans and nonhuman primates but do not cause similar disease in wild-type laboratory strains of mice unless first adapted to these animals. Although mouse adaptation has been used as a method to develop small animal models of pathogenesis, the molecular determinants associated with filovirus mouse adaptation are poorly understood. Our study demonstrates how genetic changes that accrued during mouse adaptation of the Ravn strain of Marburg virus have impacted the budding function of the viral VP40 matrix protein. Strikingly, we find impairment of mouse-adapted VP40 budding function in human but not mouse cell lines, and we correlate the impairment with an increased sensitivity of VP40 to restriction by human but not mouse tetherin and with changes in VP40 oligomerization. These data suggest that there are functional costs associated with filovirus adaptation to new hosts and implicate tetherin as a filovirus host restriction factor. PMID:25297995
Human PLC? exhibits superior fertilization potency over mouse PLC? in triggering the Ca2+ oscillations required for mammalian oocyte activation
Nomikos, Michail; Theodoridou, Maria; Elgmati, Khalil; Parthimos, Dimitris; Calver, Brian L.; Buntwal, Luke; Nounesis, George; Swann, Karl; Lai, F. Anthony
A sperm-specific phospholipase C-zeta (PLC?) is believed to play an essential role in oocyte activation during mammalian fertilization. Sperm PLC? has been shown to trigger a prolonged series of repetitive Ca2+ transients or oscillations in oocytes that precede activation. This remarkable intracellular Ca2+ signalling phenomenon is a distinctive characteristic observed during in vitro fertilization by sperm. Previous studies have notably observed an apparent differential ability of PLC? from disparate mammalian species to trigger Ca2+ oscillations in mouse oocytes. However, the molecular basis and confirmation of the apparent PLC? species difference in activity remains to be provided. In the present study, we provide direct evidence for the superior effectiveness of human PLC? relative to mouse PLC? in generating Ca2+ oscillations in mouse oocytes. In addition, we have designed and constructed a series of human/mouse PLC? chimeras to enable study of the potential role of discrete PLC? domains in conferring the enhanced Ca2+ signalling potency of human PLC?. Functional analysis of these human/mouse PLC? domain chimeras suggests a novel role of the EF-hand domain in the species-specific differences in PLC? activity. Our empirical observations are compatible with a basic mathematical model for the Ca2+ dependence of generating cytoplasmic Ca2+ oscillations in mammalian oocytes by sperm PLC?. PMID:24478462
Human PLC? exhibits superior fertilization potency over mouse PLC? in triggering the Ca(2+) oscillations required for mammalian oocyte activation.
Nomikos, Michail; Theodoridou, Maria; Elgmati, Khalil; Parthimos, Dimitris; Calver, Brian L; Buntwal, Luke; Nounesis, George; Swann, Karl; Lai, F Anthony
A sperm-specific phospholipase C-zeta (PLC?) is believed to play an essential role in oocyte activation during mammalian fertilization. Sperm PLC? has been shown to trigger a prolonged series of repetitive Ca(2+) transients or oscillations in oocytes that precede activation. This remarkable intracellular Ca(2+) signalling phenomenon is a distinctive characteristic observed during in vitro fertilization by sperm. Previous studies have notably observed an apparent differential ability of PLC? from disparate mammalian species to trigger Ca(2+) oscillations in mouse oocytes. However, the molecular basis and confirmation of the apparent PLC? species difference in activity remains to be provided. In the present study, we provide direct evidence for the superior effectiveness of human PLC? relative to mouse PLC? in generating Ca(2+) oscillations in mouse oocytes. In addition, we have designed and constructed a series of human/mouse PLC? chimeras to enable study of the potential role of discrete PLC? domains in conferring the enhanced Ca(2+) signalling potency of human PLC?. Functional analysis of these human/mouse PLC? domain chimeras suggests a novel role of the EF-hand domain in the species-specific differences in PLC? activity. Our empirical observations are compatible with a basic mathematical model for the Ca(2+) dependence of generating cytoplasmic Ca(2+) oscillations in mammalian oocytes by sperm PLC?. PMID:24478462
Mouse, but Not Human, ApoB-100 Lipoprotein Cholesterol Is a Potent Innate Inhibitor of Streptococcus pneumoniae Pneumolysin
Wade, Kristin R.; Hotze, Eileen M.; Briles, David E.; Tweten, Rodney K.
Streptococcus pneumoniae produces the pore-forming toxin pneumolysin (PLY), which is a member of the cholesterol-dependent cytolysin (CDC) family of toxins. The CDCs recognize and bind the 3?-hydroxyl group of cholesterol at the cell surface, which initiates membrane pore formation. The cholesterol transport lipoproteins, which carry cholesterol in their outer monolayer, are potential off-pathway binding targets for the CDCs and are present at significant levels in the serum and the interstitial spaces of cells. Herein we show that cholesterol carried specifically by the ApoB-100-containing lipoprotein particles (CH-ApoB-100) in the mouse, but not that carried by human or guinea pig particles, is a potent inhibitor of the PLY pore-forming mechanism. Cholesterol present in the outer monolayer of mouse ApoB-100 particles is recognized and bound by PLY, which stimulates premature assembly of the PLY oligomeric complex thereby inactivating PLY. These studies further suggest that the vast difference in the inhibitory capacity of mouse CH-ApoB-100 and that of the human and the guinea pig is due to differences in the presentation of cholesterol in the outer monolayer of their ApoB-100 particles. Therefore mouse CH-ApoB-100 represents a significant innate CDC inhibitor that is absent in humans, which may underestimate the contribution of CDCs to human disease when utilizing mouse models of disease. PMID:25188225
Novel genes in Human Asthma Based on a Mouse Model of Allergic Airway Inflammation and Human Investigations
Temesi, Gergely; Vir�g, Viktor; Hadadi, �va; Ungv�ri, Ildik�; Fodor, Lili E; Bikov, Andr�s; Nagy, Adrienne; G�lffy, Gabriella; Tam�si, Lilla; Horv�th, Ildik�; Kiss, Andr�s; Hull�m, G�bor; G�zsi, Andr�s; S�rk�zy, P�ter; Antal, P�ter; Buz�s, Edit
Purpose Based on a previous gene expression study in a mouse model of asthma, we selected 60 candidate genes and investigated their possible roles in human asthma. Methods In these candidate genes, 90 SNPs were genotyped using MassARRAY technology from 311 asthmatic children and 360 healthy controls of the Hungarian (Caucasian) population. Moreover, gene expression levels were measured by RT PCR in the induced sputum of 13 asthmatics and 10 control individuals. t-tests, chi-square tests, and logistic regression were carried out in order to assess associations of SNP frequency and expression level with asthma. Permutation tests were performed to account for multiple hypothesis testing. Results The frequency of 4 SNPs in 2 genes differed significantly between asthmatic and control subjects: SNPs rs2240572, rs2240571, rs3735222 in gene SCIN, and rs32588 in gene PPARGC1B. Carriers of the minor alleles had reduced risk of asthma with an odds ratio of 0.64 (0.51-0.80; P=7�10-5) in SCIN and 0.56 (0.42-0.76; P=1.2�10-4) in PPARGC1B. The expression levels of SCIN, PPARGC1B and ITLN1 genes were significantly lower in the sputum of asthmatics. Conclusions Three potentially novel asthma-associated genes were identified based on mouse experiments and human studies. PMID:25374748
Fibulin-2 (FBLN2): Human cDNA sequence, mRNA expression, and mapping of the gene on human and mouse chromosomes
R. Z. Zhang; T. C. Pan; Z. Y. Zhang
Fibulin-2 is a new extracellular matrix protein recently identified by characterizing mouse cDNA clones. Fibulin-2 mRNA is prominently expressed in mouse heart tissue and is present in low amounts in other tissues. In this study, the authors isolated and sequenced a 4.1-kb human fibulin-2 cDNA, which encoded a mature protein of 1157 amino acids preceded by a 27-residue signal sequence.
Isolation, characterization and propagation of mitotically active germ cells from adult mouse and human ovaries.
Woods, Dori C; Tilly, Jonathan L
Accruing evidence indicates that production of new oocytes (oogenesis) and their enclosure by somatic cells (folliculogenesis) are processes not limited to the perinatal period in mammals. Endpoints ranging from oocyte counts to genetic lineage tracing and transplantation experiments support a paradigm shift in reproductive biology involving active renewal of oocyte-containing follicles during postnatal life. The recent purification of mitotically active oocyte progenitor cells, termed female germline stem cells (fGSCs) or oogonial stem cells (OSCs), from mouse and human ovaries opens up new avenues for research into the biology and clinical utility of these cells. Here we detail methods for the isolation of mouse and human OSCs from adult ovarian tissue, cultivation of the cells after purification, and characterization of the cells before and after ex vivo expansion. The latter methods include analysis of germ cell-specific markers and in vitro oogenesis, as well as the use of intraovarian transplantation to test the oocyte-forming potential of OSCs in vivo. PMID:23598447
IL-33 induces a hypo-responsive phenotype in human and mouse mast cells
Jung, Mi-Yeon; Smr�, Daniel; Desai, Avanti; Bandara, Geethani; Ito, Tomonobu; Iwaki, Shoko; Kang, Jeong-Han; Andrade, Marcus V.; Hilderbrand, Susana C.; Brown, Jared M.; Beaven, Michael A.; Metcalfe, Dean D.; Gilfillan, Alasdair M.
SUMMARY IL-33 is elevated in afflicted tissues of patients with mast cell-dependent chronic allergic diseases. Based on its acute effects on mouse mast cells (MCs), IL-33 is thought to play a role in the pathogenesis of allergic disease through MC activation. However, the manifestations of prolonged IL-33 exposure on human MC function, which best reflect the conditions associated with chronic allergic disease, are unknown. We now find that long-term exposure of human and mouse MCs to IL-33 results in a substantial reduction of MC activation in response to antigen. This reduction required >72 h exposure to IL-33 for onset and 1�2 wk for reversion following IL-33 removal. This hypo-responsive phenotype was determined to be a consequence of MyD88-dependent attenuation of signaling processes necessary for MC activation including antigen-mediated calcium mobilization and cytoskeletal reorganization; potentially as a consequence of down-regulation of the expression of PLC?1 and Hck. These findings suggest that IL-33 may play a protective, rather than a causative role in MC activation under chronic conditions and, furthermore, reveal regulated plasticity in the MC activation phenotype. The ability to down-regulate MC activation in this manner may provide alternative approaches for treatment of MC-driven disease. PMID:23248261
Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells.
Gao, Xiugong; Sprando, Robert L; Yourick, Jeffrey J
Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72h after exposure to 0.25mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment. PMID:26006729
CD24 tracks divergent pluripotent states in mouse and human cells
Shakiba, Nika; White, Carl A.; Lipsitz, Yonatan Y.; Yachie-Kinoshita, Ayako; Tonge, Peter D; Hussein, Samer M. I.; Puri, Mira C.; Elbaz, Judith; Morrissey-Scoot, James; Li, Mira; Munoz, Javier; Benevento, Marco; Rogers, Ian M.; Hanna, Jacob H.; Heck, Albert J. R.; Wollscheid, Bernd; Nagy, Andras; Zandstra, Peter W
Reprogramming is a dynamic process that can result in multiple pluripotent cell types emerging from divergent paths. Cell surface protein expression is a particularly desirable tool to categorize reprogramming and pluripotency as it enables robust quantification and enrichment of live cells. Here we use cell surface proteomics to interrogate mouse cell reprogramming dynamics and discover CD24 as a marker that tracks the emergence of reprogramming-responsive cells, while enabling the analysis and enrichment of transgene-dependent (F-class) and -independent (traditional) induced pluripotent stem cells (iPSCs) at later stages. Furthermore, CD24 can be used to delineate epiblast stem cells (EpiSCs) from embryonic stem cells (ESCs) in mouse pluripotent culture. Importantly, regulated CD24 expression is conserved in human pluripotent stem cells (PSCs), tracking the conversion of human ESCs to more naive-like PSC states. Thus, CD24 is a conserved marker for tracking divergent states in both reprogramming and standard pluripotent culture. PMID:26076835
CD24 tracks divergent pluripotent states in mouse and human cells.
Shakiba, Nika; White, Carl A; Lipsitz, Yonatan Y; Yachie-Kinoshita, Ayako; Tonge, Peter D; Hussein, Samer M I; Puri, Mira C; Elbaz, Judith; Morrissey-Scoot, James; Li, Mira; Munoz, Javier; Benevento, Marco; Rogers, Ian M; Hanna, Jacob H; Heck, Albert J R; Wollscheid, Bernd; Nagy, Andras; Zandstra, Peter W
Reprogramming is a dynamic process that can result in multiple pluripotent cell types emerging from divergent paths. Cell surface protein expression is a particularly desirable tool to categorize reprogramming and pluripotency as it enables robust quantification and enrichment of live cells. Here we use cell surface proteomics to interrogate mouse cell reprogramming dynamics and discover CD24 as a marker that tracks the emergence of reprogramming-responsive cells, while enabling the analysis and enrichment of transgene-dependent (F-class) and -independent (traditional) induced pluripotent stem cells (iPSCs) at later stages. Furthermore, CD24 can be used to delineate epiblast stem cells (EpiSCs) from embryonic stem cells (ESCs) in mouse pluripotent culture. Importantly, regulated CD24 expression is conserved in human pluripotent stem cells (PSCs), tracking the conversion of human ESCs to more naive-like PSC states. Thus, CD24 is a conserved marker for tracking divergent states in both reprogramming and standard pluripotent culture. PMID:26076835
MMTV mouse models and the diagnostic values of MMTV-like sequences in human breast cancer
Taneja, Pankaj; Frazier, Donna P; Kendig, Robert D; Maglic, Dejan; Sugiyama, Takayuki; Kai, Fumitake; Taneja, Neetu K; Inoue, Kazushi
Mouse mammary tumor virus (MMTV) long terminal repeat (LTR)-driven transgenic mice are excellent models for breast cancer as they allow for the targeted expression of various oncogenes and growth factors in neoplastic transformation of mammary glands. Numerous MMTV-LTR-driven transgenic mouse models of breast cancer have been created in the past three decades, including MMTV-neu/ErbB2, cyclin D1, cyclin E, Ras, Myc, int-1 and c-rel. These transgenic mice develop mammary tumors with different latency, histology and invasiveness, reflecting the oncogenic pathways activated by the transgene. Recently, homologous sequences of the env gene of MMTV have been identified in approximately 40% of human breast cancers, but not in normal breast or other types of cancers, suggesting possible involvement of mammary tumor virus in human breast carcinogenesis. Accumulating evidence demonstrates the association of MMTV provirus with progesterone receptor, p53 mutations and advanced-stage breast cancer. Thus, the detection of MMTV-like sequences may have diagnostic value to predict the clinical outcome of breast cancer patients. PMID:19580428
Predominant development of mature and functional human NK cells in a novel human IL-2-producing transgenic NOG mouse.
Katano, Ikumi; Takahashi, Takeshi; Ito, Ryoji; Kamisako, Tsutomu; Mizusawa, Takuma; Ka, Yuyo; Ogura, Tomoyuki; Suemizu, Hiroshi; Kawakami, Yutaka; Ito, Mamoru
We generated a severe immunodeficient NOD/Shi-scid-IL-2R?(null) (NOG) mouse substrain expressing the transgenic human IL-2 gene (NOG-IL-2 Tg). Upon transfer of human cord blood-derived hematopoietic stem cells (HSCs), CD3(-)CD56(high)CD16(+/-) cells developed unexpectedly, predominantly in the NOG-IL-2 Tg (hu-HSC NOG-IL-2 Tg). These cells expressed various NK receptors, including NKp30, NKp44, NKp46, NKG2D, and CD94, as well as a diverse set of killer cell Ig-like receptor molecules at levels comparable to normal human NK cells from the peripheral blood, which is evidence of their maturity. They produced levels of granzyme A as high as in human peripheral blood-derived NK cells, and a considerable amount of perforin protein was detected in the plasma. Human NK cells in hu-HSC NOG-IL-2 Tg produced IFN-? upon stimulation, and IL-2, IL-15, or IL-12 treatment augmented the in vitro cytotoxicity. Inoculation of K562 leukemia cells into hu-HSC NOG-IL-2 Tg caused complete rejection of the tumor cells, whereas inoculation into hu-HSC NOG fully reconstituted with human B, T, and some NK cells did not. Moreover, when a CCR4(+) Hodgkin's lymphoma cell line was inoculated s.c. into hu-HSC NOG-IL-2 Tg, the tumor growth was significantly suppressed by treatment with a therapeutic humanized anti-CCR4 Ab (mogamulizumab), suggesting that the human NK cells in the mice exerted active Ab-dependent cellular cytotoxicity in vivo. Taken together, these data suggest that the new NOG-IL-2 Tg strain is a unique model that can be used to investigate the biological and pathological functions of human NK cells in vivo. PMID:25712215
Localization of the photoreceptor gene ROM1 to human chromosome 11 and mouse chromosome 19: Sublocalization to human 11q13 between PGA and PYGM
Bascom, R.A.; McInnes, R.R. (Univ. of Toronto (Canada)); Garcia-Heras, J.; Ledbetter, D.H.; Hsieh, C.L.; Francke, U.; Willard, F.; Jones, C.
Rom-1 is a retinal integral membrane protein that, together with the product of the human retinal degeneration slow gene (RDS), defines a photoreceptor-specific protein family. The gene for rom-1 (HGM symbol: ROM1) has been assigned to human chromosome 11 and mouse chromosome 19 by Southern blot analysis of somatic cell hybrid DNAs. ROM1 was regionally sublocalized to human 11p13-11q13 by using three mouse-human somatic cell hybrids; in situ hybridization refined the sublocalization to human 11q13. Analysis of somatic cell hybrids suggested that the most likely localization of ROM1 is in the [approximately] 2-cM interval between human PGA (human pepsinogen A) and PYGM (muscle glycogen phosphorylase). ROM1 appears to be a new member of a conserved syntenic group whose members include such genes as CD5, CD20, and OSBP (oxysterol-binding protein), on human chromosome 11 and mouse chromosome 19. Localization of the ROM1 gene will permit the examination of its linkage to hereditary retinopathies in man and mouse. 42 refs., 3 figs., 1 tab.
Genetic mapping of the human homologue (T) of mouse T(Brachyury) and a search for allele association between human T and spina bifida
Katie Morrison; Charalambos Papapetrou; John Attwood; Frans Hol; Sally A. Lynch; Anu Sampath; Ben Hamel; John Burn; Jane Sowden; David Stott; Edwin Mariman; Yvonne H. Edwards
We describe a genetic analysis of the human homo- logue (T) of the mouse T (Brachyury) gene; human T was recently cloned in our laboratory. The protein product of the T gene is a transcription factor crucial in vertebrates for the formation of normal mesoderm. T mutant Brachyury mice die in midgestation with se- vere defects in posterior mesodermal tissues;
From neural to genetic substrates of panic disorder: Insights from human and mouse studies.
Santos, M�nica; D'Amico, Davide; Dierssen, Mara
Fear is an ancestral emotion, an intrinsic defensive response present in every organism. Although fear is an evolutionarily advantageous emotion, under certain pathologies such as panic disorder it might become exaggerated and non-adaptive. Clinical and preclinical work pinpoints that changes in cognitive processes, such as perception and interpretation of environmental stimuli that rely on brain regions responsible for high-level function, are essential for the development of fear-related disorders. This review focuses on the involvement of cognitive function to fear circuitry disorders. Moreover, we address how animal models are contributing to understand the involvement of human candidate genes to pathological fear and helping achieve progress in this field. Multidisciplinary approaches that integrate human genetic findings with state of the art genetic mouse models will allow to elucidate the mechanisms underlying pathology and to develop new strategies for therapeutic targeting. PMID:25818748
Treating the Developing versus Developed Brain: Translating Preclinical Mouse and Human Studies.
Casey, B J; Glatt, Charles E; Lee, Francis S
Behaviors and underlying brain circuits show characteristic changes across the lifespan that produce sensitive windows of vulnerability and resilience to psychopathology. Understanding the developmental course of these changes may inform which treatments are best at what ages. Focusing on behavioral domains and neurobiological substrates conserved from mouse to human supports reciprocal hypothesis generation and testing that leverages the strengths of each system in understanding their development. Introducing human genetic variants into mice can further define effects of individual variation on normative development, how they contribute to risk and resilience for mental illness, and inform personalized treatment opportunities. This article emphasizes the period of adolescence, when there is a peak in the emergence of mental illness, anxiety disorders in particular. We present cross-species studies relating fear learning to anxiety across development and discuss how clinical treatments can be optimized for individuals and targeted to the biological states of the developing brain. PMID:26087163
Neurodegeneration with tau accumulation in a transgenic mouse expressing V337M human tau.
Tanemura, Kentaro; Murayama, Miyuki; Akagi, Takumi; Hashikawa, Tsutomu; Tominaga, Takashi; Ichikawa, Michinori; Yamaguchi, Haruyasu; Takashima, Akihiko
Formation of neurofibrillary tangles (NFTs) is a common neuropathological feature found in several neurodegenerative diseases, including Alzheimer's disease. We have developed a transgenic (Tg) mouse expressing mutant human tau (V337M), derived from frontotemporal dementia parkinsonism-17. V337M Tg mice revealed tau aggregations in the hippocampus, which fulfills the histological criteria for NFTs in human neurodegenerative diseases. Concurrent with the accumulation of RNA and phosphorylated tau, neurons exhibited morphological characteristics of degenerating neurons, which include a loss of microtubules, accumulation of ribosomes, plasma and nuclear membrane ruffling, and swelling of the Golgi network. Thus, mutant tau induces neuronal degeneration associated with the accumulation of RNA and phosphorylated tau. The functional consequences of this neuronal degeneration was evidenced by the reduction of hippocampal neural activity and behavioral abnormality in Tg mice. PMID:11756496
Exploiting human and mouse transcriptomic data: Identification of circadian genes and pathways influencing health.
Laing, Emma E; Johnston, Jonathan D; M�ller-Levet, Carla S; Bucca, Giselda; Smith, Colin P; Dijk, Derk-Jan; Archer, Simon N
The power of the application of bioinformatics across multiple publicly available transcriptomic data sets was explored. Using 19 human and mouse circadian transcriptomic data sets, we found that NR1D1 and NR1D2 which encode heme-responsive nuclear receptors are the most rhythmic transcripts across sleep conditions and tissues suggesting that they are at the core of circadian rhythm generation. Analyzes of human transcriptomic data show that a core set of transcripts related to processes including immune function, glucocorticoid signalling, and lipid metabolism is rhythmically expressed independently of the sleep-wake cycle. We also identify key transcripts associated with transcription and translation that are disrupted by sleep manipulations, and through network analysis identify putative mechanisms underlying the adverse health outcomes associated with sleep disruption, such as diabetes and cancer. Comparative bioinformatics applied to existing and future data sets will be a powerful tool for the identification of core circadian- and sleep-dependent molecules. PMID:25772847
Immunochemical analysis of the N-acetyl hexosaminidases in human-mouse hybrids made using a double selective system
D. M. Swallow; E. Solomon; L. Pajunen
A human-mouse hybrid, DUR 4 (Solomon et al., 1976), containing a human X\\/15 translocation chromosome and also chromosome 5, among other human chromosomes, was used in a double selection system to obtain hybrids of four different types: X\\/15+ 5+, X\\/15� 5+, X\\/15+ 5, and X\\/15� 5�. Standard positive and negative selection systems were used for the X chromosome, and negative
Expression and in vivo rescue of human ABCC6 disease-causing mutants in mouse liver.
Le Saux, Olivier; F�l�p, Krisztina; Yamaguchi, Yukiko; Ili�s, Attila; Szab�, Zal�n; Brampton, Christopher N; Pomozi, Viola; Husz�r, Krisztina; Ar�nyi, Tam�s; V�radi, Andr�s
Loss-of-function mutations in ABCC6 can cause chronic or acute forms of dystrophic mineralization described in disease models such as pseudoxanthoma elasticum (OMIM 26480) in human and dystrophic cardiac calcification in mice. The ABCC6 protein is a large membrane-embedded organic anion transporter primarily found in the plasma membrane of hepatocytes. We have established a complex experimental strategy to determine the structural and functional consequences of disease-causing mutations in the human ABCC6. The major aim of our study was to identify mutants with preserved transport activity but failure in intracellular targeting. Five missense mutations were investigated: R1138Q, V1298F, R1314W, G1321S and R1339C. Using in vitro assays, we have identified two variants; R1138Q and R1314W that retained significant transport activity. All mutants were transiently expressed in vivo, in mouse liver via hydrodynamic tail vein injections. The inactive V1298F was the only mutant that showed normal cellular localization in liver hepatocytes while the other mutants showed mostly intracellular accumulation indicating abnormal trafficking. As both R1138Q and R1314W displayed endoplasmic reticulum localization, we tested whether 4-phenylbutyrate (4-PBA), a drug approved for clinical use, could restore their intracellular trafficking to the plasma membrane in MDCKII and mouse liver. The cellular localization of R1314W was significantly improved by 4-PBA treatment, thus potentially rescuing its physiological function. Our work demonstrates the feasibility of the in vivo rescue of cellular maturation of some ABCC6 mutants in physiological conditions very similar to the biology of the fully differentiated human liver and could have future human therapeutic application. PMID:21935449
Expression and In Vivo Rescue of Human ABCC6 Disease-Causing Mutants in Mouse Liver
Le Saux, Olivier; F�l�p, Krisztina; Yamaguchi, Yukiko; Ili�s, Attila; Szab�, Zal�n; Brampton, Christopher N.; Pomozi, Viola; Husz�r, Krisztina; Ar�nyi, Tam�s; V�radi, Andr�s
Loss-of-function mutations in ABCC6 can cause chronic or acute forms of dystrophic mineralization described in disease models such as pseudoxanthoma elasticum (OMIM 26480) in human and dystrophic cardiac calcification in mice. The ABCC6 protein is a large membrane-embedded organic anion transporter primarily found in the plasma membrane of hepatocytes. We have established a complex experimental strategy to determine the structural and functional consequences of disease-causing mutations in the human ABCC6. The major aim of our study was to identify mutants with preserved transport activity but failure in intracellular targeting. Five missense mutations were investigated: R1138Q, V1298F, R1314W, G1321S and R1339C. Using in vitro assays, we have identified two variants; R1138Q and R1314W that retained significant transport activity. All mutants were transiently expressed in vivo, in mouse liver via hydrodynamic tail vein injections. The inactive V1298F was the only mutant that showed normal cellular localization in liver hepatocytes while the other mutants showed mostly intracellular accumulation indicating abnormal trafficking. As both R1138Q and R1314W displayed endoplasmic reticulum localization, we tested whether 4-phenylbutyrate (4-PBA), a drug approved for clinical use, could restore their intracellular trafficking to the plasma membrane in MDCKII and mouse liver. The cellular localization of R1314W was significantly improved by 4-PBA treatment, thus potentially rescuing its physiological function. Our work demonstrates the feasibility of the in vivo rescue of cellular maturation of some ABCC6 mutants in physiological conditions very similar to the biology of the fully differentiated human liver and could have future human therapeutic application. PMID:21935449
Current humanized mouse models for studying human immunology and HIV1 immuno-pathogenesis
LiGuo Zhang; Eric Meissner; JianZhu Chen; LiShan Su
A robust animal model for �hypothesis-testing\\/mechanistic� research in human immunology and immuno-pathology should meet the\\u000a following criteria. First, it has well-studied hemato-lymphoid organs and target cells similar to those of humans. Second,\\u000a the human pathogens establish infection and lead to relevant diseases. Third, it is genetically inbred and can be manipulated\\u000a via genetic, immunological and pharmacological means. Many human-tropic pathogens
Chromosomally unstable mouse tumours have genomic alterations similar to diverse human cancers
Maser, Richard S.; Choudhury, Bhudipa; Campbell, Peter J.; Feng, Bin; Wong, Kwok-Kin; Protopopov, Alexei; O�Neil, Jennifer; Gutierrez, Alejandro; Ivanova, Elena; Perna, Ilana; Lin, Eric; Mani, Vidya; Jiang, Shan; McNamara, Kate; Zaghlul, Sara; Edkins, Sarah; Stevens, Claire; Brennan, Cameron; Martin, Eric S.; Wiedemeyer, Ruprecht; Kabbarah, Omar; Nogueira, Cristina; Histen, Gavin; Aster, Jon; Mansour, Marc; Duke, Veronique; Foroni, Letizia; Fielding, Adele K.; Goldstone, Anthony H.; Rowe, Jacob M.; Wang, Yaoqi A.; Look, A. Thomas; Stratton, Michael R.; Chin, Lynda; Futreal, P. Andrew; DePinho, Ronald A.
Highly rearranged and mutated cancer genomes present major challenges in the identification of pathogenetic events driving the neoplastic transformation process. Here we engineered lymphoma-prone mice with chromosomal instability to assess the usefulness of mouse models in cancer gene discovery and the extent of cross-species overlap in cancer-associated copy number aberrations. Along with targeted re-sequencing, our comparative oncogenomic studies identified FBXW7 and PTEN to be commonly deleted both in murine lymphomas and in human T-cell acute lymphoblastic leukaemia/lymphoma (T-ALL). The murine cancers acquire widespread recurrent amplifications and deletions targeting loci syntenic to those not only in human T-ALL but also in diverse human haematopoietic, mesenchymal and epithelial tumours. These results indicate that murine and human tumours experience common biological processes driven by orthologous genetic events in their malignant evolution. The highly concordant nature of genomic events encourages the use of genomically unstable murine cancer models in the discovery of biological driver events in the human oncogenome. PMID:17515920
Transgenic mouse strains as platforms for the successful discovery and development of human therapeutic monoclonal antibodies.
Green, Larry L
Transgenic mice have yielded seven of the ten currently-approved human antibody drugs, making them the most successful platform for the discovery of fully human antibody therapeutics. The use of the in vivo immune system helps drive this success by taking advantage of the natural selection process that produces antibodies with desirable characteristics. Appropriately genetically-engineered mice act as robust engines for the generation of diverse repertoires of affinity- matured fully human variable regions with intrinsic properties necessary for successful antibody drug development including high potency, specificity, manufacturability, solubility and low risk of immunogenicity. A broad range of mAb drug targets are addressable in these mice, comprising both secreted and transmembrane targets, including membrane multi-spanning targets, as well as human target antigens that share high sequence identity with their mouse orthologue. Transgenic mice can routinely yield antibodies with sub-nanomolar binding affinity for their antigen, with lead candidate mAbs frequently possessing affinities for binding to their target of less than 100 picomolar, without requiring any ex vivo affinity optimization. While the originator transgenic mice platforms are no longer broadly available, a new generation of transgenic platforms is in development for discovery of the next wave of human therapeutic antibodies. PMID:23978036
Production of MPS VII mouse (Gustm(hE540A�mE536A)Sly) doubly tolerant to human and mouse ?-glucuronidase
Tomatsu, Shunji; Orii, Koji O.; Vogler, Carole; Grubb, Jeffrey H.; Snella, Elizabeth M.; Gutierrez, Monica; Dieter, Tatiana; Holden, Christopher C.; Sukegawa, Kazuko; Orii, Tadao; Kondo, Naomi; Sly, William S.
Mucopolysaccharidosis VII (MPS VII, Sly syndrome) is an autosomal recessive lysosomal storage disease caused by ?-glucuronidase (GUS) deficiency. A naturally occurring mouse model of that disease has been very useful for studying experimental approaches to therapy. However, immune responses can complicate evaluation of the long-term benefits of enzyme replacement or gene therapy delivered to adult MPS VII mice. To make this model useful for studying the long-term effectiveness and side effects of experimental therapies delivered to adult mice, we developed a new MPS VII mouse model, which is tolerant to both human and murine GUS. To achieve this, we used homologous recombination to introduce simultaneously a human cDNA transgene expressing inactive human GUS into intron 9 of the murine Gus gene and a targeted active site mutation (E536A) into the adjacent exon 10. When the heterozygote products of germline transmission were bred to homozygosity, the homozygous mice expressed no GUS enzyme activity but expressed inactive human GUS protein highly and were tolerant to immune challenge with human enzyme. Expression of the mutant murine Gus gene was reduced to about 10% of normal levels, but the inactive murine GUS enzyme also conferred tolerance to murine GUS. This MPS VII mouse model should be useful to evaluate therapeutic responses in adult mice receiving repetitive doses of enzyme or mice receiving gene therapy as adults. Heterozygotes expressed only 9.5�26% of wild-type levels of murine GUS instead of the expected 50%, indicating a dominant-negative effect of the mutant enzyme monomers on the activity of GUS tetramers in different tissues. Corrective gene therapy in this model should provide high enough levels of expression of normal GUS monomers to overcome the dominant negative effect of mutant monomers on newly synthesized GUS tetramers in most tissues. PMID:12700165
Comparative analysis of temporal and dose-dependent TCDD-elicited gene expression in human, mouse, and rat primary hepatocytes.
Forgacs, Agnes L; Dere, Edward; Angrish, Michelle M; Zacharewski, Timothy R
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-elicited time- and dose-dependent differential gene expression was compared in human, mouse, and rat primary hepatocytes. Comprehensive time course (10 nM TCDD or dimethyl sulfoxide vehicle control for 1, 2, 4, 8, 12, 24, and 48h) studies identified 495, 2305, and 711 differentially expressed orthologous genes in human, mouse, and rat hepatocytes, respectively. However, only 16 orthologs were differentially expressed across all three species, with the majority of orthologs exhibiting species-specific expression (399 human, 2097 mouse, and 533 rat), consistent with species-specific expression reported in other in vitro and in vivo comparative studies. TCDD also elicited the dose-dependent induction of 397 human, 100 mouse, and 443 rat genes at 12h and 615 human, 426 mouse, and 314 rat genes at 24h. Comparable EC50 values were obtained for AhR battery genes including Cyp1a1 (0.1 nM human, 0.05 nM mouse, 0.08 nM rat at 24h) and Tiparp (0.97 nM human, 0.63 nM mouse, 0.14 nM rat at 12h). Overrepresented functions and pathways included amino acid metabolism in humans, immune response in mice, and energy homeostasis in rats. Differentially expressed genes functionally associated with lipid transport, processing, and metabolism were overrepresented in all three species but exhibited species-specific expression consistent with the induction of hepatic steatosis in mice but not in rats following a single oral gavage of TCDD. Furthermore, human primary hepatocytes showed lipid accumulation following 48h of treatment with TCDD, suggesting that AhR-mediated steatosis in mice more closely resembles human hepatic fat accumulation compared with that in rats. Collectively, these results suggest that species-specific gene expression profiles mediate the species-specific effects of TCDD despite the conservation of the AhR and its signaling mechanism. PMID:23418086
The absence of a human-specific ribosomal DNA transcription factor leads to nucleolar dominance in mouse greater than human hybrid cells.
Miesfeld, R; Sollner-Webb, B; Croce, C; Arnheim, N
The basis for nucleolar dominance in mouse-human cell hybrids which contained all of the mouse chromosomes but an incomplete set of human chromosomes (M greater than H) was examined at the molecular level. S1 mapping data showed that these cells had the expected levels of steady-state rRNA transcribed from mouse ribosomal gene (rDNA) transcription units but undetectable levels of rRNA derived from the human rDNA transcription templates that are also present. RNA polymerase I-dependent, cell-free transcription extracts were made from three hybrid lines and were found to be capable of transcribing cloned rDNA templates of mouse but not human origin. Partially purified human factors required for rDNA transcription in vitro were added to the M greater than H extracts. One fraction with almost no RNA polymerase I activity conferred on these hybrid cell extracts the ability to transcribe a human rDNA template. These rescue experiments suggested that this required human-specific rDNA transcription factor(s) was effectively absent from the lines we examined and could account for nucleolar dominance in M greater than H hybrid cells. Images PMID:6095060
Sequences in Influenza A Virus PB2 Protein That Determine Productive Infection for an Avian Influenza Virus in Mouse and Human Cell Lines
Yao, Yongxiu; Mingay, Louise J.; McCauley, John W.; Barclay, Wendy S.
Reverse genetics was used to analyze the host range of two avian influenza viruses which differ in their ability to replicate in mouse and human cells in culture. Engineered viruses carrying sequences encoding amino acids 362 to 581 of PB2 from a host range variant productively infect mouse and human cells. PMID:11333926
The Mouse Hoxd13 spdh Mutation, a Polyalanine Expansion Similar to Human Type II Synpolydactyly (SPD), Disrupts the Function but Not the Expression of Other Hoxd Genes
Sylvia Bruneau; Kenneth R. Johnson; Masakazu Yamamoto; Atsushi Kuroiwa; Denis Duboule
Polyalanine expansion in the human HOXD13 gene induces synpolydactyly (SPD), an inherited congenital limb malformation. A mouse model was isolated, which showed a spontaneous alanine expansion due to a 21-bp duplication at the corresponding place in the mouse gene. This mutation (synpolydactyly homolog, spdh), when homozygous, causes malformations in mice similar to those seen in affected human patients. We have
Development of a nude mouse model to study human sebaceous gland physiology and pathophysiology.
Petersen, M J; Zone, J J; Krueger, G G
Study of human sebaceous gland physiology and pathophysiology is limited by lack of an adequate animal model. This study was designed to develop an animal model using human face skin grafted onto the nude mouse to study human sebaceous glands. Full-thickness human face skin was grafted onto 60 adult male nude mice. 4 wk after grafting, androgens, which are known to stimulate sebaceous glands, were administered to test the system. Androgens were administered to 21 animals by implanted catheters that were filled with testosterone (T) or dihydrotestosterone (DHT). Empty catheters were implanted in 15 control animals. Graft biopsies and blood for androgen levels were obtained at time 1 (pre-catheter) and time 2 (26 d after catheter implantation). Three assessments were made on each biopsy: sebaceous gland volume, using an image analyzing computer; sebaceous cell size; and sebaceous gland labeling index. 29 mice completed the study through time 2. In the androgen-treated group, T levels (nanogram per milliliter) five times increased to 4.92 +/- 0.35, and DHT levels (nanogram per milliliter) increased 50 times to 16.70. In the androgen-treated group, sebaceous gland volume (micron 3 X 10(-3) increased from 896 +/- 194 to 3,233 +/- 754 (P less than 0.001), sebaceous cell area (micron 2) increased from 167 +/- 12 to 243 +/- 19 (P less than 0.001), and labeling index (percentage) increased from 2.7 +/- 0.7 to 6.4 +/- 0.9 (P less than 0.01). In the control group, sebaceous gland volume fell from 1,070 +/- 393 to 417 +/- 99 (NS), sebaceous cell size remained the same, and the labeling index fell from 5.1 +/- 1.9 to 3.2 +/- 1.1. After androgen administration, Halowax N-34, a known comedogen, or its vehicle, was applied to 15 grafts for 2-6 wk. Twice as many microcomedones were seen in the Halowax-treated grafts, compared with vehicle-treated grafts at the end of this time period. No visible comedones were produced. This study demonstrated that: (a) human sebaceous glands can be successfully transplanted and studied on the nude mouse; (b) after androgen stimulation, sebaceous gland volume, cell size, and labeling index increase; (c) microcomedones can be produced in the human skin grafts by the application of a comedogenic substance. Thus, this model demonstrates significant potential for the future study of human sebaceous gland physiology and pathology. Images PMID:6237122
Sox10 Expressing Cells in the Lateral Wall of the Aged Mouse and Human Cochlea
Hao, Xinping; Xing, Yazhi; Moore, Michael W.; Zhang, Jianning; Han, Demin; Schulte, Bradley A.; Dubno, Judy R.; Lang, Hainan
Age-related hearing loss (presbycusis) is a common human disorder, affecting one in three Americans aged 60 and over. Previous studies have shown that presbyacusis is associated with a loss of non-sensory cells in the cochlear lateral wall. Sox10 is a transcription factor crucial to the development and maintenance of neural crest-derived cells including some non-sensory cell types in the cochlea. Mutations of the Sox10 gene are known to cause various combinations of hearing loss and pigmentation defects in humans. This study investigated the potential relationship between Sox10 gene expression and pathological changes in the cochlear lateral wall of aged CBA/CaJ mice and human temporal bones from older donors. Cochlear tissues prepared from young adult (1�3 month-old) and aged (2�2.5 year-old) mice, and human temporal bone donors were examined using quantitative immunohistochemical analysis and transmission electron microscopy. Cells expressing Sox10 were present in the stria vascularis, outer sulcus and spiral prominence in mouse and human cochleas. The Sox10+ cell types included marginal and intermediate cells and outer sulcus cells, including those that border the scala media and those extending into root processes (root cells) in the spiral ligament. Quantitative analysis of immunostaining revealed a significant decrease in the number of Sox10+ marginal cells and outer sulcus cells in aged mice. Electron microscopic evaluation revealed degenerative alterations in the surviving Sox10+ cells in aged mice. Strial marginal cells in human cochleas from donors aged 87 and older showed only weak immunostaining for Sox10. Decreases in Sox10 expression levels and a loss of Sox10+ cells in both mouse and human aged ears suggests an important role of Sox10 in the maintenance of structural and functional integrity of the lateral wall. A loss of Sox10+ cells may also be associated with a decline in the repair capabilities of non-sensory cells in the aged ear. PMID:24887110
Sox10 expressing cells in the lateral wall of the aged mouse and human cochlea.
Hao, Xinping; Xing, Yazhi; Moore, Michael W; Zhang, Jianning; Han, Demin; Schulte, Bradley A; Dubno, Judy R; Lang, Hainan
Age-related hearing loss (presbycusis) is a common human disorder, affecting one in three Americans aged 60 and over. Previous studies have shown that presbyacusis is associated with a loss of non-sensory cells in the cochlear lateral wall. Sox10 is a transcription factor crucial to the development and maintenance of neural crest-derived cells including some non-sensory cell types in the cochlea. Mutations of the Sox10 gene are known to cause various combinations of hearing loss and pigmentation defects in humans. This study investigated the potential relationship between Sox10 gene expression and pathological changes in the cochlear lateral wall of aged CBA/CaJ mice and human temporal bones from older donors. Cochlear tissues prepared from young adult (1-3 month-old) and aged (2-2.5 year-old) mice, and human temporal bone donors were examined using quantitative immunohistochemical analysis and transmission electron microscopy. Cells expressing Sox10 were present in the stria vascularis, outer sulcus and spiral prominence in mouse and human cochleas. The Sox10(+) cell types included marginal and intermediate cells and outer sulcus cells, including those that border the scala media and those extending into root processes (root cells) in the spiral ligament. Quantitative analysis of immunostaining revealed a significant decrease in the number of Sox10(+) marginal cells and outer sulcus cells in aged mice. Electron microscopic evaluation revealed degenerative alterations in the surviving Sox10(+) cells in aged mice. Strial marginal cells in human cochleas from donors aged 87 and older showed only weak immunostaining for Sox10. Decreases in Sox10 expression levels and a loss of Sox10(+) cells in both mouse and human aged ears suggests an important role of Sox10 in the maintenance of structural and functional integrity of the lateral wall. A loss of Sox10(+) cells may also be associated with a decline in the repair capabilities of non-sensory cells in the aged ear. PMID:24887110
A Novel Mouse Model for Stable Engraftment of a Human Immune System and Human Hepatocytes
Strick-Marchand, Helene; Duss�aux, Mathilde; Darche, Sylvie; Huntington, Nicholas D.; Legrand, Nicolas; Masse-Ranson, Guillemette; Corcuff, Erwan; Ahodantin, James; Weijer, Kees; Spits, Hergen; Kremsdorf, Dina; Di Santo, James P.
Hepatic infections by hepatitis B virus (HBV), hepatitis C virus (HCV) and Plasmodium parasites leading to acute or chronic diseases constitute a global health challenge. The species tropism of these hepatotropic pathogens is restricted to chimpanzees and humans, thus model systems to study their pathological mechanisms are severely limited. Although these pathogens infect hepatocytes, disease pathology is intimately related to the degree and quality of the immune response. As a first step to decipher the immune response to infected hepatocytes, we developed an animal model harboring both a human immune system (HIS) and human hepatocytes (HUHEP) in BALB/c Rag2-/- IL-2R?c-/- NOD.sirpa uPAtg/tg mice. The extent and kinetics of human hepatocyte engraftment were similar between HUHEP and HIS-HUHEP mice. Transplanted human hepatocytes were polarized and mature in vivo, resulting in 20�50% liver chimerism in these models. Human myeloid and lymphoid cell lineages developed at similar frequencies in HIS and HIS-HUHEP mice, and splenic and hepatic compartments were humanized with mature B cells, NK cells and na�ve T cells, as well as monocytes and dendritic cells. Taken together, these results demonstrate that HIS-HUHEP mice can be stably (> 5 months) and robustly engrafted with a humanized immune system and chimeric human liver. This novel HIS-HUHEP model provides a platform to investigate human immune responses against hepatotropic pathogens and to test novel drug strategies or vaccine candidates. PMID:25782010
Human and mouse trace amine-associated receptor 1 have distinct pharmacology towards endogenous monoamines and imidazoline receptor ligands.
Hu, Liaoyuan A; Zhou, Tian; Ahn, Jinwoo; Wang, Shuli; Zhou, Julia; Hu, Yi; Liu, Qingyun
TAARs (trace amine-associated receptors) are G-protein-coupled receptors that respond to low abundance, endogenous amines such as tyramine and tryptamine, and represent potential targets for neuropsychiatric diseases. However, some members of this receptor subfamily either have no ligand identified or remain difficult to express and characterize using recombinant systems. In the present paper we report the successful expression of human and mouse TAAR1, and the characterization of their responses to various natural and synthetic agonists. In HEK (human embryonic kidney)-293/CRE-bla cells, mouse TAAR1 showed a robust response to trace amines as measured using either a cAMP assay or a beta-lactamase reporter assay, whereas human TAAR1 showed a weaker, but still measurable, response. When certain fragments of human TAAR1 were replaced with the corresponding regions of mouse TAAR1, the chimaeric receptor showed a much stronger response in cAMP production. Examination of a series of agonists on these receptors revealed that the human and the chimaeric receptor are almost identical in pharmacology, but distinct from the mouse receptor. We also screened small libraries of pharmacologically active agents on TAAR1 and identified a series of synthetic agonists, some of which are also ligands of the enigmatic imidazoline receptor. The findings of the present study not only shed light on the pharmacological species difference of TAAR1, but also raise new possibilities about the mechanism of some of the imidazoline-related agents. PMID:19725810
NSDL National Science Digital Library
In this activity, learners explore the concept of how engineering solved the problem of human/computer interface. Learners disassemble a mouse and explore the movement on the X/Y axis that determines mouse positioning. Learners explore design enhancements to the mouse over time, and as a team of "engineers" add further enhancements to current mouse design.
Pre- and postexposure efficacy of fully human antibodies against Spike protein in a novel humanized mouse model of MERS-CoV infection
Pascal, Kristen E.; Coleman, Christopher M.; Mujica, Alejandro O.; Kamat, Vishal; Badithe, Ashok; Fairhurst, Jeanette; Hunt, Charleen; Strein, John; Berrebi, Alexander; Sisk, Jeanne M.; Matthews, Krystal L.; Babb, Robert; Chen, Gang; Lai, Ka-Man V.; Huang, Tammy T.; Olson, William; Yancopoulos, George D.; Stahl, Neil; Frieman, Matthew B.; Kyratsous, Christos A.
Traditional approaches to antimicrobial drug development are poorly suited to combatting the emergence of novel pathogens. Additionally, the lack of small animal models for these infections hinders the in vivo testing of potential therapeutics. Here we demonstrate the use of the VelocImmune technology (a mouse that expresses human antibody-variable heavy chains and ? light chains) alongside the VelociGene technology (which allows for rapid engineering of the mouse genome) to quickly develop and evaluate antibodies against an emerging viral disease. Specifically, we show the rapid generation of fully human neutralizing antibodies against the recently emerged Middle East Respiratory Syndrome coronavirus (MERS-CoV) and development of a humanized mouse model for MERS-CoV infection, which was used to demonstrate the therapeutic efficacy of the isolated antibodies. The VelocImmune and VelociGene technologies are powerful platforms that can be used to rapidly respond to emerging epidemics. PMID:26124093
Localization of Shaw-related K+ channel genes on mouse and human chromosomes.
Haas, M; Ward, D C; Lee, J; Roses, A D; Clarke, V; D'Eustachio, P; Lau, D; Vega-Saenz de Miera, E; Rudy, B
Four related genes, Shaker, Shab, Shaw, and Shal, encode voltage-gated K+ channels in Drosophila. Multigene subfamilies corresponding to each of these Drosophila genes have been identified in rodents and primates; this suggests that the four genes are older than the common ancestor of present-day insects and mammals and that the expansion of each into a family occurred before the divergence of rodents and primates. In order to define these evolutionary relationships more precisely and to facilitate the search for mammalian candidate K+ channel gene mutations, we have mapped members of the Shaw-homologous gene family in humans and mice. Fluorescence in situ hybridization analysis of human metaphase chromosomes mapped KCNC2 (KShIIIA, KV3.2) and KCNC3 (KShIIID, KV3.3) to Chromosome (Chr) 19q13.3-q13.4. Inheritance patterns of DNA restriction fragment length variants in recombinant inbred strains of mice placed the homologous mouse genes on distal Chr 10 near Ms15-8 and Mdm-1. The mouse Kcnc1 (KShIIIB, NGK2-KV4, KV3.1) gene mapped to Chr7 near Tam-1. These results are consistent with the hypothesis that the generation of the mammalian KCNC gene family included both duplication events to generate family members in tandem arrays (KCNC2, KCNC3) and dispersion of family members to unlinked chromosomal sites (KCNC1). The KNCN2 and KCNC3 genes define a new synteny group between humans and mice. PMID:8111118
Antifibrotic Effects of Antioxidant N-Acetylcysteine in a Mouse Model of Human Hypertrophic Cardiomyopathy Mutation
Marian, Ali J.; Senthil, Vinitha; Chen, Suet N.; Lombardi, Raffaella
OBJECTIVES The objective was to determine the effects of antioxidant N-acetylcysteine (NAC) on reversal and attenuation of established interstitial fibrosis in the cardiac troponin T (cTnT) mouse model of human hypertrophic cardiomyopathy (HCM) mutation. BACKGROUND Interstitial fibrosis is a characteristic pathological feature of HCM and a risk factor for sudden cardiac death. The cTnT-Q92 transgenic mice, generated by cardiac-restricted expression of human HCM mutation, show a two- to four-fold increase in interstitial fibrosis. METHODS We randomized the cTnT-Q92 mice to treatment with a placebo or NAC (250, 500, or 1,000 mg/kg/day) and included non-transgenic mice as controls (N = 5 to 13 per group). We performed echocardiography before and 24 weeks after therapy, followed by histologic and molecular characterization. RESULTS There were no significant differences in the baseline characteristics of the groups. Treatment with NAC reduced myocardial concentrations of malondialdehyde and 4-hydroxy-2(E)-nonenal, markers of oxidative stress, by 40%. Collagen volume fractions comprised 1.94 � 0.76% of the myocardium in non-transgenic, 6.2 � 1.65% in the placebo, and 1.56 � 0.98% in the NAC (1,000 mg/kg/day) groups (p < 0.001). Expression levels of Col1a1 and Col1a2 were also reduced significantly, as were levels of phosphorylated but not total p44/42, p38, and c-Jun NH2-terminal kinase. Levels of oxidized mitochondrial and nuclear DNA were not significantly different. CONCLUSIONS Treatment with NAC reduced myocardial oxidative stress, stress-responsive signaling kinases, and fibrosis in a mouse model of HCM. The potential beneficial effects of NAC in reversal of cardiac phenotype in human HCM, the most common cause of sudden cardiac death in the young, merits investigation. PMID:16487852
Evaluation of Depigmenting Activity by 8-Hydroxydaidzein in Mouse B16 Melanoma Cells and Human Volunteers
Tai, Sorgan Shou-Ku; Lin, Ching-Gong; Wu, Mon-Han; Chang, Te-Sheng
In our previous study, 8-hydroxydaidzein (8-OHDe) was demonstrated to be a potent and unique suicide substrate of mushroom tyrosinase. In this study, the compound was evaluated for in vitro cellular tyrosinase and melanogenesis inhibitory activities in mouse B16 melanoma cells and for in vivo skin-whitening activity in human volunteers. Tyrosinase activity and melanogenesis in the cell culture incubated with 10 ?M of 8-OHDe were decreased to 20.1% and 51.8% of control, respectively, while no obvious cytotoxicity was observed in this concentration. In contrast, a standard tyrosinase inhibitor, kojic acid, showed 69.9% and 71.3% of control in cellular tyrosinase and melanogenesis activity, respectively, at a concentration as high as 100 ?M. Hence, 8-OHDe exhibited more than an inhibitory effects on melanin production in B16 cells 10-fold stronger than kojic acid. In addition, when a cream containing 4% 8-OHDe was applied to human skin in an in vivo study, significant increases in the dL*-values were observed after three weeks. Moreover, the increase in the dL*-values after 8-week treatment with 4% 8-OHDe (from ?0.57 to 1.94) is stronger than those of 2% 8-OHDe treatment (from 0.26 to 0.94) and 2% ascorbic acid-2-glucoside treatment (from 0.07 to 1.54). From the results of the study, it was concluded that 8-OHDe, the potent suicide substrate of mushroom tyrosinase, has depigmenting activities in both mouse melanoma cells and in human volunteers. Thus, the compound has significant potential for use in cosmetics as a skin-whitening ingredient. PMID:20057943
Significant expansion of the REST/NRSF cistrome in human versus mouse embryonic stem cells: potential implications for neural development.
Rockowitz, Shira; Zheng, Deyou
Recent studies have employed cross-species comparisons of transcription factor binding, reporting significant regulatory network 'rewiring' between species. Here, we address how a transcriptional repressor targets and regulates neural genes differentially between human and mouse embryonic stem cells (ESCs). We find that the transcription factor, Repressor Element 1 Silencing Transcription factor (REST; also called neuron restrictive silencer factor) binds to a core group of ?1200 syntenic genomic regions in both species, with these conserved sites highly enriched with co-factors, selective histone modifications and DNA hypomethylation. Genes with conserved REST binding are enriched with neural functions and more likely to be upregulated upon REST depletion. Interestingly, we identified twice as many REST peaks in human ESCs compared to mouse ESCs. Human REST cistrome expansion involves additional peaks in genes targeted by REST in both species and human-specific gene targets. Genes with expanded REST occupancy in humans are enriched for learning or memory functions. Analysis of neurological disorder associated genes reveals that Amyotrophic Lateral Sclerosis and oxidative stress genes are particularly enriched with human-specific REST binding. Overall, our results demonstrate that there is substantial rewiring of human and mouse REST cistromes, and that REST may have human-specific roles in brain development and functions. PMID:25990720
Completely Humanizing Prolactin Rescues Infertility in Prolactin Knockout Mice and Leads to Human Prolactin Expression in Extrapituitary Mouse Tissues
Christensen, Heather R.; Murawsky, Michael K.; Horseman, Nelson D.; Willson, Tara A.
A variety of fundamental differences have evolved in the physiology of the human and rodent prolactin (PRL) systems. The PRL gene in humans and other primates contains an alternative promoter, 5.8 kbp upstream of the pituitary transcription start site, which drives expression of PRL in �extrapituitary� tissues, where PRL is believed to exert local, or paracrine, actions. Several of these extrapituitary PRL tissues serve a reproductive function (eg, mammary gland, decidua, prostate, etc), consistent with the hypothesis that local PRL production may be involved in, and required for, normal reproductive physiology in primates. Rodent research models have generated significant findings regarding the role of PRL in reproduction. Specifically, disruption (knockout) of either the PRL gene or its receptor causes profound female reproductive defects at several levels (ovaries, preimplantation endometrium, mammary glands). However, the rodent PRL gene differs significantly from the human, most notably lacking the alternative promoter. Understanding of the physiological regulation and function of extrapituitary PRL has been limited by the absence of a readily accessible experimental model, because the rodent PRL gene does not contain the alternative promoter. To overcome these limitations, we have generated mice that have been �humanized� with regard to the structural gene and tissue expression of PRL. Here, we present the characterization of these animals, demonstrating that the human PRL transgene is responsive to known physiological regulators both in vitro and in vivo. More importantly, the expression of the human PRL transgene is able to rescue the reproductive defects observed in mouse PRL knockout (mPRL?) females, validating their usefulness in studying the function or regulation of this hormone in a manner that is relevant to human physiology. PMID:24029242
Analysis of PRICKLE1 in human cleft palate and mouse development demonstrates rare and common variants involved in human malformations
Yang, Tian; Jia, Zhonglin; Bryant-Pike, Whitney; Chandrasekhar, Anand; Murray, Jeffrey C; Fritzsch, Bernd; Bassuk, Alexander G
Palate development is shaped by multiple molecular signaling pathways, including the Wnt pathway. In mice and humans, mutations in both the canonical and noncanonical arms of the Wnt pathway manifest as cleft palate, one of the most common human birth defects. Like the palate, numerous studies also link different Wnt signaling perturbations to varying degrees of limb malformation; for example, shortened limbs form in mutations of Ror2,Vangl2looptail and, in particular, Wnt5a. We recently showed the noncanonical Wnt/planar cell polarity (PCP) signaling molecule Prickle1 (Prickle like 1) also stunts limb growth in mice. We now expanded these studies to the palate and show that Prickle1 is also required for palate development, like Wnt5a and Ror2. Unlike in the limb, the Vangl2looptail mutation only aggravates palate defects caused by other mutations. We screened Filipino cleft palate patients and found PRICKLE1 variants, both common and rare, at an elevated frequency. Our results reveal that in mice and humans PRICKLE1 directs palate morphogenesis; our results also uncouple Prickle1 function from Vangl2 function. Together, these findings suggest mouse and human palate development is guided by PCP-Prickle1 signaling that is probably not downstream of Vangl2. PMID:24689077
Immortalization of human lymphocytes by transfection with DNA from mouse L929 cytoplasts
Abken, H.; Buetzler, C.; Willecke, K.
Transfection of human peripheral blood lymphocytes with DNA from mouse L929 cytoplasts induced proliferation of lymphocytes and the formation of B and T cell-derived cell lines with apparently unlimited growth potential. The cell lines could be grown in serum-containing media as well as in chemically defined serum-free media, have a nearly normal human karyotype, did not form colonies in soft-agar medium, and were not tumorigenic after injection into nude mice. For immortalization of human lymphocytes DNA from L929 cytoplasts was 100-fold more efficient than L929 nuclear DNA. The ability of cytoplast DNA to immortalize lymphocytes could be consecutively transferred by using total cellular DNA from primary or secondary transfectants. Circular or linear mitochondrial DNA of L929 cells did not lead to immortilization of human lymphocytes. Since DNA with immortalizing activity could be isolated from cytoplasts, the Hirt supernatant, and a mitochondria-depleted cytoplasmic fraction of L929 cells. The authors conclude that the immortalizing DNA is located extramitochondrially in the cytoplasm of L929 cells.
Somatic mutations of the ?-catenin gene are frequent in mouse and human hepatocellular carcinomas
Coste, Alix de La; Romagnolo, B�atrice; Billuart, Pierre; Renard, Claire-Ang�lique; Buendia, Marie-Annick; Soubrane, Olivier; Fabre, Monique; Chelly, Jamel; Beldjord, Cherif; Kahn, Axel; Perret, Christine
Hepatocellular carcinoma (HCC) is the major primary malignant tumor in the human liver, but the molecular changes leading to liver cell transformation remain largely unknown. The Wnt-?-catenin pathway is activated in colon cancers and some melanoma cell lines, but has not yet been investigated in HCC. We have examined the status of the ?-catenin gene in different transgenic mouse lines of HCC obtained with the oncogenes c-myc or H-ras. Fifty percent of the hepatic tumors in these transgenic mice had activating somatic mutations within the ?-catenin gene similar to those found in colon cancers and melanomas. These alterations in the ?-catenin gene (point mutations or deletions) lead to a disregulation of the signaling function of ?-catenin and thus to carcinogenesis. We then analyzed human HCCs and found similar mutations in eight of 31 (26%) human liver tumors tested and in HepG2 and HuH6 hepatoma cells. The mutations led to the accumulation of ?-catenin in the nucleus. Thus alterations in the ?-catenin gene frequently are selected for during liver tumorigenesis and suggest that disregulation of the Wnt-?-catenin pathway is a major event in the development of HCC in humans and mice. PMID:9671767
Checkpoint-apoptosis uncoupling in human and mouse embryonic stem cells: a source of karyotpic instability
Guo, Ying; Lee, Man Ryul; Kim, Min-Kyoung; Han, Myung-Kwan; Shibayama, Hirohiko; Fukuda, Seiji; Yoder, Mervin C.; Pelus, Louis M.
Karyotypic abnormalities in cultured embryonic stem cells (ESCs), especially near-diploid aneuploidy, are potential obstacles to ESC use in regenerative medicine. Events causing chromosomal abnormalities in ESCs may be related to events in tumor cells causing chromosomal instability (CIN) in human disease. However, the underlying mechanisms are unknown. Using multiparametric permeabilized-cell flow cytometric analysis, we found that the mitotic-spindle checkpoint, which helps maintain chromosomal integrity during all cell divisions, functions in human and mouse ESCs, but does not initiate apoptosis as it does in somatic cells. This allows an unusual tolerance to polyploidy resulting from failed mitosis, which is common in rapidly proliferating cell populations and which is reduced to near-diploid aneuploidy, which is also common in human neoplastic disease. Checkpoint activation in ESC-derived early-differentiated cells results in robust apoptosis without polyploidy/aneuploidy similar to that in somatic cells. Thus, the spindle checkpoint is �uncoupled� from apoptosis in ESCs and is a likely source of karyotypic abnormalities. This natural behavior of ESCs to tolerate/survive varying degrees of ploidy change could complicate genome-reprogramming studies and stem-cell plasticity studies, but could also reveal clues about the mechanisms of CIN in human tumors. PMID:17289813
Mouse Models of Diet-Induced Nonalcoholic Steatohepatitis Reproduce the Heterogeneity of the Human Disease
Machado, Mariana Verdelho; Michelotti, Gregory Alexander; Xie, Guanhua; de Almeida, Thiago Pereira; Boursier, Jerome; Bohnic, Brittany; Guy, Cynthia D.; Diehl, Anna Mae
Background and aims Non-alcoholic steatohepatitis (NASH), the potentially progressive form of nonalcoholic fatty liver disease (NAFLD), is the pandemic liver disease of our time. Although there are several animal models of NASH, consensus regarding the optimal model is lacking. We aimed to compare features of NASH in the two most widely-used mouse models: methionine-choline deficient (MCD) diet and Western diet. Methods Mice were fed standard chow, MCD diet for 8 weeks, or Western diet (45% energy from fat, predominantly saturated fat, with 0.2% cholesterol, plus drinking water supplemented with fructose and glucose) for 16 weeks. Liver pathology and metabolic profile were compared. Results The metabolic profile associated with human NASH was better mimicked by Western diet. Although hepatic steatosis (i.e., triglyceride accumulation) was also more severe, liver non-esterified fatty acid content was lower than in the MCD diet group. NASH was also less severe and less reproducible in the Western diet model, as evidenced by less liver cell death/apoptosis, inflammation, ductular reaction, and fibrosis. Various mechanisms implicated in human NASH pathogenesis/progression were also less robust in the Western diet model, including oxidative stress, ER stress, autophagy deregulation, and hedgehog pathway activation. Conclusion Feeding mice a Western diet models metabolic perturbations that are common in humans with mild NASH, whereas administration of a MCD diet better models the pathobiological mechanisms that cause human NAFLD to progress to advanced NASH. PMID:26017539
Restorative effect of hair follicular dermal cells on injured human hair follicles in a mouse model.
Yamao, Mikaru; Inamatsu, Mutsumi; Okada, Taro; Ogawa, Yuko; Ishida, Yuji; Tateno, Chise; Yoshizato, Katsutoshi
No model is available for examining whether in vivo-damaged human hair follicles (hu-HFs) are rescued by transplanting cultured hu-HF dermal cells (dermal papilla and dermal sheath cells). Such a model might be valuable for examining whether in vivo-damaged hu-HFs such as miniaturized hu-HFs in androgenic alopecia are improvable by auto-transplanting hu-HF dermal cells. In this study, we first developed mice with humanized skin composed of hu-keratinocytes and hu-dermal fibroblasts. Then, a 'humanized scalp model mouse' was generated by transplanting hu-scalp HFs into the humanized skin. To demonstrate the usability of the model, the lower halves of the hu-HFs in the model were amputated in situ, and cultured hu-HF dermal cells were injected around the amputated area. The results demonstrated that the transplanted cells contributed to the restoration of the damaged HFs. This model could be used to explore clinically effective technologies for hair restoration therapy by autologous cell transplantation. PMID:25557326
Pathway-Specific Engineered Mouse Allograft Models Functionally Recapitulate Human Serous Epithelial Ovarian Cancer
Szabova, Ludmila; Bupp, Sujata; Kamal, Muhaymin; Householder, Deborah B.; Hernandez, Lidia; Schlomer, Jerome J.; Baran, Maureen L.; Yi, Ming; Stephens, Robert M.; Annunziata, Christina M.; Martin, Philip L.; Van Dyke, Terry A.
The high mortality rate from ovarian cancers can be attributed to late-stage diagnosis and lack of effective treatment. Despite enormous effort to develop better targeted therapies, platinum-based chemotherapy still remains the standard of care for ovarian cancer patients, and resistance occurs at a high rate. One of the rate limiting factors for translation of new drug discoveries into clinical treatments has been the lack of suitable preclinical cancer models with high predictive value. We previously generated genetically engineered mouse (GEM) models based on perturbation of Tp53 and Rb with or without Brca1 or Brca2 that develop serous epithelial ovarian cancer (SEOC) closely resembling the human disease on histologic and molecular levels. Here, we describe an adaptation of these GEM models to orthotopic allografts that uniformly develop tumors with short latency and are ideally suited for routine preclinical studies. Ovarian tumors deficient in Brca1 respond to treatment with cisplatin and olaparib, a PARP inhibitor, whereas Brca1-wild type tumors are non-responsive to treatment, recapitulating the relative sensitivities observed in patients. These mouse models provide the opportunity for evaluation of effective therapeutics, including prediction of differential responses in Brca1-wild type and Brca1�deficient tumors and development of relevant biomarkers. PMID:24748377
Systematic Identification of cis-Regulatory Sequences Active in Mouse and Human Embryonic Stem Cells
Gaspar-Maia, Alexandre; Li, Hao; Ramalho-Santos, Miguel
Understanding the transcriptional regulation of pluripotent cells is of fundamental interest and will greatly inform efforts aimed at directing differentiation of embryonic stem (ES) cells or reprogramming somatic cells. We first analyzed the transcriptional profiles of mouse ES cells and primordial germ cells and identified genes upregulated in pluripotent cells both in vitro and in vivo. These genes are enriched for roles in transcription, chromatin remodeling, cell cycle, and DNA repair. We developed a novel computational algorithm, CompMoby, which combines analyses of sequences both aligned and non-aligned between different genomes with a probabilistic segmentation model to systematically predict short DNA motifs that regulate gene expression. CompMoby was used to identify conserved overrepresented motifs in genes upregulated in pluripotent cells. We show that the motifs are preferentially active in undifferentiated mouse ES and embryonic germ cells in a sequence-specific manner, and that they can act as enhancers in the context of an endogenous promoter. Importantly, the activity of the motifs is conserved in human ES cells. We further show that the transcription factor NF-Y specifically binds to one of the motifs, is differentially expressed during ES cell differentiation, and is required for ES cell proliferation. This study provides novel insights into the transcriptional regulatory networks of pluripotent cells. Our results suggest that this systematic approach can be broadly applied to understanding transcriptional networks in mammalian species. PMID:17784790
Pathway-specific engineered mouse allograft models functionally recapitulate human serous epithelial ovarian cancer.
Szabova, Ludmila; Bupp, Sujata; Kamal, Muhaymin; Householder, Deborah B; Hernandez, Lidia; Schlomer, Jerome J; Baran, Maureen L; Yi, Ming; Stephens, Robert M; Annunziata, Christina M; Martin, Philip L; Van Dyke, Terry A; Weaver Ohler, Zoe; Difilippantonio, Simone
The high mortality rate from ovarian cancers can be attributed to late-stage diagnosis and lack of effective treatment. Despite enormous effort to develop better targeted therapies, platinum-based chemotherapy still remains the standard of care for ovarian cancer patients, and resistance occurs at a high rate. One of the rate limiting factors for translation of new drug discoveries into clinical treatments has been the lack of suitable preclinical cancer models with high predictive value. We previously generated genetically engineered mouse (GEM) models based on perturbation of Tp53 and Rb with or without Brca1 or Brca2 that develop serous epithelial ovarian cancer (SEOC) closely resembling the human disease on histologic and molecular levels. Here, we describe an adaptation of these GEM models to orthotopic allografts that uniformly develop tumors with short latency and are ideally suited for routine preclinical studies. Ovarian tumors deficient in Brca1 respond to treatment with cisplatin and olaparib, a PARP inhibitor, whereas Brca1-wild type tumors are non-responsive to treatment, recapitulating the relative sensitivities observed in patients. These mouse models provide the opportunity for evaluation of effective therapeutics, including prediction of differential responses in Brca1-wild type and Brca1-deficient tumors and development of relevant biomarkers. PMID:24748377
Recombinant Human Epidermal Growth Factor Accelerates Recovery of Mouse Small Intestinal Mucosa After Radiation Damage
Lee, Kang Kyoo [Department of Radiation Oncology, University of Wonkwang School of Medicine, Iksan (Korea, Republic of); Jo, Hyang Jeong [Department of Pathology, University of Wonkwang School of Medicine, Iksan (Korea, Republic of); Hong, Joon Pio [Department of Plastic and Reconstructive Surgery, University of Ulsan College of Medicine, Asan Medical Center, Seoul (Korea, Republic of); Lee, Sang-wook [Department of Radiation Oncology, University of Ulsan College of Medicine, Asan Medical Center, Seoul (Korea, Republic of)], E-mail: firstname.lastname@example.com; Sohn, Jung Sook [Vestibulocochlear Research Center, University of Wonkwang School of Medicine, Iksan (Korea, Republic of); Moon, Soo Young [Department of Radiation Oncology, University of Ulsan College of Medicine, Asan Medical Center, Seoul (Korea, Republic of); Yang, Sei Hoon; Shim, Hyeok [Department of Internal Medicine, University of Wonkwang School of Medicine, Iksan (Korea, Republic of); Lee, Sang Ho [Department of Radiology, Iksan General Hospital, Iksan (Korea, Republic of); Ryu, Seung-Hee [Department of Radiation Oncology, University of Ulsan College of Medicine, Asan Medical Center, Seoul (Korea, Republic of); Moon, Sun Rock [Department of Radiation Oncology, University of Wonkwang School of Medicine, Iksan (Korea, Republic of)
Purpose: To determine whether systemically administered recombinant human epidermal growth factor (rhEGF) accelerates the recovery of mouse small intestinal mucosa after irradiation. Methods and Materials: A mouse mucosal damage model was established by administering radiation to male BALB/c mice with a single dose of 15 Gy applied to the abdomen. After irradiation, rhEGF was administered subcutaneously at various doses (0.04, 0.2, 1.0, and 5.0 mg/kg/day) eight times at 2- to 3-day intervals. The evaluation methods included histologic changes of small intestinal mucosa, change in body weight, frequency of diarrhea, and survival rate. Results: The recovery of small intestinal mucosa after irradiation was significantly improved in the mice treated with a high dose of rhEGF. In the mice that underwent irradiation without rhEGF treatment, intestinal mucosal ulceration, mucosal layer damage, and severe inflammation occurred. The regeneration of villi was noticeable in mice treated with more than 0.2 mg/kg rhEGF, and the villi recovered fully in mice given more than 1 mg/kg rhEGF. The frequency of diarrhea persisting for more than 3 days was significantly greater in the radiation control group than in the rhEGF-treated groups. Conclusions: Systemic administration of rhEGF accelerates recovery from mucosal damage induced by irradiation. We suggest that rhEGF treatment shows promise for the reduction of small intestinal damage after irradiation.
Development of humanized mouse models to study human malaria parasite infection
Vaughan, Ashley M; Kappe, Stefan HI; Ploss, Alexander; Mikolajczak, Sebastian A
Malaria is a disease caused by infection with Plasmodium parasites that are transmitted by mosquito bite. Five different species of Plasmodium infect humans with severe disease, but human malaria is primarily caused by Plasmodium falciparum. The burden of malaria on the developing world is enormous, and a fully protective vaccine is still elusive. One of the biggest challenges in the quest for the development of new antimalarial drugs and vaccines is the lack of accessible animal models to study P. falciparum infection because the parasite is restricted to the great apes and human hosts. Here, we review the current state of research in this field and provide an outlook of the development of humanized small animal models to study P. falciparum infection that will accelerate fundamental research into human parasite biology and could accelerate drug and vaccine design in the future. PMID:22568719
Development of a Transgenic Mouse with R124H Human TGFBI Mutation Associated with Granular Corneal Dystrophy Type 2
Yasuda, Miyuki; Hatou, Shin; Inagaki, Emi; Ogawa, Yoko; Tsubota, Kazuo; Shimmura, Shigeto
Purpose To investigate the phenotype and predisposing factors of a granular corneal dystrophy type 2 transgenic mouse model. Methods Human TGFBI cDNA with R124H mutation was used to make a transgenic mouse expressing human protein (TGFBIR124H mouse). Reverse transcription PCR (RT-PCR) was performed to analyze TGFBIR124H expression. A total of 226 mice including 23 homozygotes, 106 heterozygotes and 97 wild-type mice were examined for phenotype. Affected mice were also examined by histology, immunohistochemistry and electron microcopy. Results RT-PCR confirmed the expression of TGFBIR124H in transgenic mice. Corneal opacity defined as granular and lattice deposits was observed in 45.0% of homozygotes, 19.4% of heterozygotes. The incidence of corneal opacity was significantly higher in homozygotes than in heterozygotes (p = 0.02). Histology of affected mice was similar to histology of human disease. Lesions were Congo red and Masson Trichrome positive, and were observed as a deposit of amorphous material by electron microscopy. Subepithelial stroma was also stained with thioflavin T and LC3, a marker of autophagy activation. The incidence of corneal opacity was higher in aged mice in each group. Homozygotes were not necessarily more severe than heterozygotes, which deffers from human cases. Conclusions We established a granular corneal dystrophy type 2 mouse model caused by R124H mutation of human TGFBI. Although the phenotype of this mouse model is not equivalent to that in humans, further studies using this model may help elucidate the pathophysiology of this disease. PMID:26197481
Novel Insights into Embryonic Stem Cell Self-Renewal Revealed Through Comparative Human and Mouse Systems Biology Networks
Dowell, Karen G.; Simons, Allen K.; Bai, Hao; Kell, Braden; Wang, Zack Z.; Yun, Kyuson; Hibbs, Matthew A.
Embryonic stem cells (ESCs), characterized by their ability to both self-renew and differentiate into multiple cell lineages, are a powerful model for biomedical research and developmental biology. Human and mouse ESCs share many features, yet have distinctive aspects, including fundamental differences in the signaling pathways and cell cycle controls that support self-renewal. Here, we explore the molecular basis of human ESC self-renewal using Bayesian network machine learning to integrate cell-type-specific, high-throughput data for gene function discovery. We integrated high-throughput ESC data from 83 human studies (~1.8 million data points collected under 1100 conditions) and 62 mouse studies (~2.4 million data points collected under 1085 conditions) into separate human and mouse predictive networks focused on ESC self-renewal to analyze shared and distinct functional relationships among protein-coding gene orthologs. Computational evaluations show that these networks are highly accurate, literature validation confirms their biological relevance, and RT-PCR validation supports our predictions. Our results reflect the importance of key regulatory genes known to be strongly associated with self-renewal and pluripotency in both species (e.g. POU5F1, SOX2, and NANOG), identify metabolic differences between species (e.g. threonine metabolism), clarify differences between human and mouse ESC developmental signaling pathways (e.g. LIF-activated JAK/STAT in mouse; NODAL/ACTIVIN-A-activated FGF in human), and reveal many novel genes and pathways predicted to be functionally associated with self-renewal in each species. These interactive networks are available online at www.StemSight.org for stem cell researchers to develop new hypotheses, discover potential mechanisms involving sparsely annotated genes, and prioritize genes of interest for experimental validation. PMID:24307629
Mouse p53-Deficient Cancer Models as Platforms for Obtaining Genomic Predictors of Human Cancer Clinical Outcomes
Due�as, Marta; Santos, Mirentxu; Aranda, Juan F.; Bielza, Concha; Mart�nez-Cruz, Ana B.; Lorz, Corina; Taron, Miquel; Ciruelos, Eva M.; Rodr�guez-Peralto, Jos� L.; Mart�n, Miguel; Larra�aga, Pedro; Dahabreh, Jubrail; Stathopoulos, George P.; Rosell, Rafael; Paramio, Jes�s M.; Garc�a-Escudero, Ram�n
Mutations in the TP53 gene are very common in human cancers, and are associated with poor clinical outcome. Transgenic mouse models lacking the Trp53 gene or that express mutant Trp53 transgenes produce tumours with malignant features in many organs. We previously showed the transcriptome of a p53-deficient mouse skin carcinoma model to be similar to those of human cancers with TP53 mutations and associated with poor clinical outcomes. This report shows that much of the 682-gene signature of this murine skin carcinoma transcriptome is also present in breast and lung cancer mouse models in which p53 is inhibited. Further, we report validated gene-expression-based tests for predicting the clinical outcome of human breast and lung adenocarcinoma. It was found that human patients with cancer could be stratified based on the similarity of their transcriptome with the mouse skin carcinoma 682-gene signature. The results also provide new targets for the treatment of p53-defective tumours. PMID:22880004
Immunohistochemical localization of zona pellucida proteins ZPA, ZPB and ZPC in human, cynomolgus monkey and mouse ovaries
U. Eberspaecher; A. Becker; P. Bringmann; L. van der Merwe; P. Donner
The zona pellucida of mammalian oocytes plays an important role in binding and activation of sperm cells during the molecular events leading to fertilization. The genes coding for the three zona pellucida glycoproteins ZPA, ZPB, and ZPC of various species including mouse, dog, and human have been cloned and sequenced by several groups. However, it has remained a matter of
Conversion of Mouse and Human Fibroblasts into Functional Spinal Motor Neurons
Son, Esther Y.; Ichida, Justin K.; Wainger, Brian J.; Toma, Jeremy S.; Rafuse, Victor F.; Woolf, Clifford J.; Eggan, Kevin
SUMMARY The mammalian nervous system is composed of a multitude of distinct neuronal subtypes, each with its own phenotype and differential sensitivity to degenerative disease. Although specific neuronal types can be isolated from rodent embryos or engineered from stem cells for translational studies, transcription factor mediated reprogramming might provide a more direct route to their generation. Here we report that the forced expression of select transcription factors is sufficient to convert mouse and human fibroblasts into induced motor neurons (iMNs). iMNs displayed a morphology, gene expression signature, electrophysiology, synaptic functionality, in vivo engraftment capacity and sensitivity to degenerative stimuli, similar to embryo-derived motor neurons. We show that the converting fibroblasts do not transit through a proliferative neural progenitor state, and thus form bona fide motor neurons via a route distinct from embryonic development. Our findings demonstrate that fibroblasts can be converted directly into a specific differentiated and functional neural subtype, the spinal motor neuron. PMID:21852222
Neuronal migration disorders in humans and in mouse models - an overview
Copp, Andrew J.; Harding, Brian N.
The spectrum of neuronal migration disorders (NMD) in humans encompasses developmental brain defects with a range of clinical and pathological features. A simple classification distinguishes agyria/pachygyria, heterotopia, polymicrogyria and cortical dysplasia as distinct clinico-pathological entities. Many of these conditions are associated with intractable epilepsy. When considering the pathogenesis of NMD, a critical developmental process is the migration of neuroblasts along the processes of radial glia during the formation of the layered structure of the cerebral cortex. In addition, faulty cytodifferentiation and programmed cell death play important roles in the generation of dysplasias and heterotopias respectively. A number of genes have been identified that participate in the regulation of neuronal migration. Mouse models, in which these genes are mutated, provide insight into the developmental pathways that underlie normal and abnormal neuronal migration. PMID:10515161
Mapping of the taurine transporter gene to mouse chromosome 6 and to the short arm of human chromosome 3
Patel, A.; Uhl, G.R.; Gregor, P. [National Inst. of Health, Baltimore, MD (United States)] [and others] [National Inst. of Health, Baltimore, MD (United States); and others
Transport proteins have essential functions in the uptake of neurotransmitters and neuromodulators. We have mapped the gene encoding the taurine transporter, Taut, to the central region of mouse chromosome 6. Analysis of a cross segregating the neurological mutant mnd2 excluded Taut as a candidate gene for this closely linked mutation. To map the human taurine transporter gene, TAUT, a sequence-tagged site (STS) corresponding to the 3{prime} untranslated region of the human cDNA was developed. TAUT was assigned to human chromosome 3 by typing this STS on a panel of somatic cell hybrids. Further analysis of a hybrid panel containing defined deletions of chromosome 3 suggested that TAUT maps to 3p21-p25. These data extend a conserved linkage group on mouse chromosome 6 and human chromosome 3p. Deletion of TAUT might contribute to some phenotypic features of the 3p{sup -} syndrome. 32 refs., 3 figs.
Subepidermal Blistering Induced by Human Autoantibodies to BP180 Requires Innate Immune Players in a Humanized Bullous Pemphigoid Mouse Model
Liu, Zhi; Sui, Wen; Zhao, Minglang; Li, Zhuowei; Li, Ning; Thresher, Randy; Giudice, George J.; Fairley, Janet A.; Sitaru, Cassian; Zillikens, Detlef; Ning, Gang; Marinkovich, Peter; Diaz, Luis A.
Bullous pemphigoid (BP) is a cutaneous autoimmune inflammatory disease associated with subepidermal blistering and autoantibodies against BP180, a transmembrane collagen and major component of the hemidesmosome. Numerous inflammatory cells infiltrate the upper dermis in BP. IgG autoantibodies in BP fix complement and target multiple BP180 epitopes that are highly clustered within a non-collagen linker domain, termed NC16A. Anti-BP180 antibodies induce BP in mice. In this study, we generated a humanized mouse strain, in which the murine BP180NC14A is replaced with the homologous human BP180NC16A epitope cluster region. We show that the humanized NC16A (NC16A+/+) mice injected with anti-BP180NC16A autoantibodies develop BP-like subepidermal blisters. The F(ab?)2 fragments of pathogenic IgG fail to activate complement cascade and are no longer pathogenic. The NC16A+/+ mice pretreated with mast cell activation blocker or depleting of complement or neutrophils become resistant to BP. These findings suggest that the humoral response in BP critically depends on innate immune system players. PMID:18922680
Subepidermal blistering induced by human autoantibodies to BP180 requires innate immune players in a humanized bullous pemphigoid mouse model.
Liu, Zhi; Sui, Wen; Zhao, Minglang; Li, Zhuowei; Li, Ning; Thresher, Randy; Giudice, George J; Fairley, Janet A; Sitaru, Cassian; Zillikens, Detlef; Ning, Gang; Marinkovich, M Peter; Diaz, Luis A
Bullous pemphigoid (BP) is a cutaneous autoimmune inflammatory disease associated with subepidermal blistering and autoantibodies against BP180, a transmembrane collagen and major component of the hemidesmosome. Numerous inflammatory cells infiltrate the upper dermis in BP. IgG autoantibodies in BP fix complement and target multiple BP180 epitopes that are highly clustered within a non-collagen linker domain, termed NC16A. Anti-BP180 antibodies induce BP in mice. In this study, we generated a humanized mouse strain, in which the murine BP180NC14A is replaced with the homologous human BP180NC16A epitope cluster region. We show that the humanized NC16A (NC16A+/+) mice injected with anti-BP180NC16A autoantibodies develop BP-like subepidermal blisters. The F(ab')(2) fragments of pathogenic IgG fail to activate the complement cascade and are no longer pathogenic. The NC16A+/+ mice pretreated with mast cell activation blocker or depleted of complement or neutrophils become resistant to BP. These findings suggest that the humoral response in BP critically depends on innate immune system players. PMID:18922680
Development and characterization of a novel mouse line humanized for the intestinal peptide transporter PEPT1.
Hu, Yongjun; Xie, Yehua; Wang, Yuqing; Chen, Xiaomei; Smith, David E
The proton-coupled oligopeptide transporter PEPT1 (SLC15A1) is abundantly expressed in the small intestine, but not colon, of mammals and found to mediate the uptake of di/tripeptides and peptide-like drugs from the intestinal lumen. However, species differences have been observed in both the expression (and localization) of PEPT1 and its substrate affinity. With this in mind, the objectives of this study were to develop a humanized PEPT1 mouse model (huPEPT1) and to characterize hPEPT1 expression and functional activity in the intestines. Thus, after generating huPEPT1 mice in animals previously nulled for mouse Pept1, phenotypic, PCR, and immunoblot analyses were performed, along with in situ single-pass intestinal perfusion and in vivo oral pharmacokinetic studies with a model dipeptide, glycylsarcosine (GlySar). Overall, the huPEPT1 mice had normal survival rates, fertility, litter size, gender distribution, and body weight. There was no obvious behavioral or pathological phenotype. The mRNA and protein profiles indicated that huPEPT1 mice had substantial PEPT1 expression in all regions of the small intestine (i.e., duodenum, jejunum, and ileum) along with low but measurable expression in both proximal and distal segments of the colon. In agreement with PEPT1 expression, the in situ permeability of GlySar in huPEPT1 mice was similar to but lower than wildtype animals in small intestine, and greater than wildtype mice in colon. However, a species difference existed in the in situ transport kinetics of jejunal PEPT1, in which the maximal flux and Michaelis constant of GlySar were reduced 7-fold and 2- to 4-fold, respectively, in huPEPT1 compared to wildtype mice. Still, the in vivo function of intestinal PEPT1 appeared fully restored (compared to Pept1 knockout mice) as indicated by the nearly identical pharmacokinetics and plasma concentration-time profiles following a 5.0 nmol/g oral dose of GlySar to huPEPT1 and wildtype mice. This study reports, for the first time, the development and characterization of mice humanized for PEPT1. This novel transgenic huPEPT1 mouse model should prove useful in examining the role, relevance, and regulation of PEPT1 in diet and disease, and in the drug discovery process. PMID:25148225
Bisected, complex N-glycans and galectins in mouse mammary tumor progression and human breast cancer
Miwa, Hazuki E; Koba, Wade R; Fine, Eugene J; Giricz, Orsi; Kenny, Paraic A; Stanley, Pamela
Bisected, complex N-glycans on glycoproteins are generated by the glycosyltransferase MGAT3 and cause reduced cell surface binding of galectins. Previously, we showed that MGAT3 reduces growth factor signaling and retards mammary tumor progression driven by the Polyoma middle T antigen (PyMT) expressed in mammary epithelium under the mouse mammary tumor virus (MMTV) promoter. However, the penetrance of the tumor phenotype became variable in mixed FVB/N and C57BL/6 female mice and we therefore investigated a congenic C57BL/6 Mgat3?/?/MMTV-PyMT model. In the absence of MGAT3, C57BL/6 Mgat3?/?/MMTV-PyMT females exhibited accelerated tumor appearance and increased tumor burden, glucose uptake in tumors and lung metastasis. Nevertheless, activation of extracellular signal-regulated kinase (ERK)1/2 or protein kinase B (AKT) was reduced in ?20-week C57BL/6 MMTV-PyMT tumors lacking MGAT3. Activation of focal adhesion kinase (FAK), protein tyrosine kinase Src, and p38 mitogen-activated protein kinase were similar to that of controls. All the eight mouse galectin genes were expressed in mammary tumors and tumor epithelial cells (TECs), but galectin-2 and -12 were not detected by western analysis in tumors, and galectin-7 was not detected in 60% of the TEC lines. From microarray data reported for human breast cancers, at least 10 galectin and 7 N-glycan N-acetylglucosaminyl (GlcNAc)-transferase (MGAT) genes are expressed in tumor tissue, and expression often varies significantly between different breast cancer subtypes. Thus, in summary, while MGAT3 and bisected complex N-glycans retard mouse mammary tumor progression, genetic background may modify this effect; identification of key galectins that promote mammary tumor progression in mice is not straightforward because all the eight galectin genes are expressed; and high levels of MGAT3, galectin-4, -8, -10, -13 and -14 transcripts correlate with better relapse-free survival in human breast cancer. PMID:24037315
The Human Blue Opsin Promoter Directs Transgene Expression in Short-Wave Cones and Bipolar Cells in the Mouse Retina
J. Chen; C. L. Tucker; B. Woodford; A. Szel; J. Lem; A. Gianella-Borradori; M. I. Simon; E. Bogenmann
Transgenic mouse lines were generated using either 3.8 or 1.1 kb of 5' upstream flanking sequence from the human blue opsin gene fused to the lacZ or human growth hormone reporter gene. Mice were analyzed for appropriate cell-specific and developmental expression patterns. In 13 independently derived lines of animals, transgene expression was limited to photoreceptor and inner nuclear layer cells.
Sleeping Beauty mutagenesis in a mouse medulloblastoma model defines networks that discriminate between human molecular subgroups.
Genovesi, Laura A; Ng, Ching Ging; Davis, Melissa J; Remke, Marc; Taylor, Michael D; Adams, David J; Rust, Alistair G; Ward, Jerrold M; Ban, Kenneth H; Jenkins, Nancy A; Copeland, Neal G; Wainwright, Brandon J
The Sleeping Beauty (SB) transposon mutagenesis screen is a powerful tool to facilitate the discovery of cancer genes that drive tumorigenesis in mouse models. In this study, we sought to identify genes that functionally cooperate with sonic hedgehog signaling to initiate medulloblastoma (MB), a tumor of the cerebellum. By combining SB mutagenesis with Patched1 heterozygous mice (Ptch1(lacZ/+)), we observed an increased frequency of MB and decreased tumor-free survival compared with Ptch1(lacZ/+) controls. From an analysis of 85 tumors, we identified 77 common insertion sites that map to 56 genes potentially driving increased tumorigenesis. The common insertion site genes identified in the mutagenesis screen were mapped to human orthologs, which were used to select probes and corresponding expression data from an independent set of previously described human MB samples, and surprisingly were capable of accurately clustering known molecular subgroups of MB, thereby defining common regulatory networks underlying all forms of MB irrespective of subgroup. We performed a network analysis to discover the likely mechanisms of action of subnetworks and used an in vivo model to confirm a role for a highly ranked candidate gene, Nfia, in promoting MB formation. Our analysis implicates candidate cancer genes in the deregulation of apoptosis and translational elongation, and reveals a strong signature of transcriptional regulation that will have broad impact on expression programs in MB. These networks provide functional insights into the complex biology of human MB and identify potential avenues for intervention common to all clinical subgroups. PMID:24167280
Expression of human sequences related to those of mouse mammary tumor virus.
Franklin, G C; Chretien, S; Hanson, I M; Rochefort, H; May, F E; Westley, B R
Sequences related to those of the mouse mammary tumor virus (MuMTV) genome have been cloned from human DNA by screening a library prepared from the DNA of a human breast cancer cell line with MuMTV gag-pol DNA. Nine distinct groups of (MuMTV-related) sequences were identified among 100 lambda recombinants by cross-hybridization experiments with subcloned fragments containing gag-pol-related DNA. The largest group, of 64 recombinants, contains the MuMTV-related sequences cloned by others. The other eight groups contain MuMTV-related sequences that have not been described previously. The gag-pol regions of one recombinant from each of the nine groups were hybridized to RNA prepared from five human breast cancer cell lines, from placenta, and from two cell lines derived from other malignancies. RNAs were detected by probes for seven of the groups. The RNAs ranged in size from 1.2 to 12 kilobases. Probes for six of the groups detected large RNAs that could represent transcripts of full-length proviral DNA. Two of the probes detected RNA in one breast cancer cell line only. Most of the RNAs were detected in more than one cell line. Images PMID:2831381
Structure and expression of the human and mouse T4 genes.
Maddon, P J; Molineaux, S M; Maddon, D E; Zimmerman, K A; Godfrey, M; Alt, F W; Chess, L; Axel, R
The T4 molecule may serve as a T-cell receptor recognizing molecules on the surface of specific target cells and also serves as the receptor for the human immunodeficiency virus. To define the mechanisms of interaction of T4 with the surface of antigen-presenting cells as well as with human immunodeficiency virus, we have further analyzed the sequence, structure, and expression of the human and mouse T4 genes. T4 consists of an extracellular segment comprised of a leader sequence followed by four tandem variable-joining (VJ)-like domains, a transmembrane domain, and a cytoplasmic segment. The structural domains of the T4 protein deduced from amino acid sequence are precisely reflected in the intron-exon organization of the gene. Analysis of the expression of the T4 gene indicates that T4 RNA is expressed not only in T lymphocytes, but in B cells, macrophages, and granulocytes. T4 is also expressed in a developmentally regulated manner in specific regions of the brain. It is, therefore, possible that T4 plays a more general role in mediating cell recognition events that are not restricted to the cellular immune response. Images PMID:3501122
Structure and expression of the human and mouse T4 genes
Maddon, P.J.; Molineaux, S.M.; Maddon, D.F.; Zimmerman, K.A.; Godfrey, M.; Alt, F.W.; Chess, L.; Axel, R.
The T4 molecule may serve as a T-cell receptor recognizing molecules on the surface of specific target cells and also serves as the receptor for the human immunodeficiency virus. To define the mechanisms of interaction of T4 with the surface of antigen-presenting cells as well as with human immunodeficiency virus, the authors have further analyzed the sequence, structure, and expression of the human and mouse T4 genes. T4 consists of an extracellular segment comprised of a leader sequence followed by four tandem variable-joining (VJ)-like domains, a transmembrane domain, and A cytoplasmic segment. The structural domains of the T4 protein deduced from amino acid sequence are precisely reflected in the intron-exon organization of the gene. Analysis of the expression of the T4 gene indicates that T4 RNA is expressed not only in T lymphocytes, but in B cells, macrophages, and granulocytes. T4 is also expressed in a developmentally regulated manner in specific regions of the brain. It is, therefore, possible that T4 plays a more general role in mediating cell recognition events that are not restricted to the cellular immune response.
Comparative metabolism of geranyl nitrile and citronellyl nitrile in mouse, rat, and human hepatocytes.
Kemper, Raymond A; Nabb, Diane L; Gannon, Shawn A; Snow, Timothy A; Api, Anne Marie
Geranyl nitrile (GN) and citronellyl nitrile (CN) are fragrance components used in consumer and personal care products. Differences in the clastogenicity of these two terpenes are postulated to result from differential biotransformation, presumably involving the conjugated nitrile moiety. The metabolic clearance and biotransformation of GN and CN were compared in primary hepatocytes from mice, rats, and humans. For determination of intrinsic clearance, GN and CN were incubated with hepatocytes in sealed vials, and the headspace was sampled periodically by solid-phase microextraction and analyzed by gas chromatography/mass spectrometry. For metabolite identification, GN and CN were incubated with hepatocytes from each species for 60 min, and reaction mixtures were extracted and analyzed by mass spectroscopy. Both GN and CN were rapidly metabolized in hepatocytes from all species (T1/2, 0.7-11.6 min). Within a species, intrinsic clearance was similar for both compounds and increased in the order human < rat < mouse. Major common pathways for biotransformation of GN and CN involved 1) epoxidation of the 6-alkenyl moiety followed by conjugation with glutathione, 2) hydroxylation of the terminal methyl group(s) followed by direct conjugation with glucuronic acid in rodents or further oxidation to the corresponding acid in human cells, and 3) hydroxylation of the allylic C5 position. No evidence for either phase I or phase II metabolism of the conjugated nitrile moiety was obtained. Thus, the presumed metabolic basis for differences in genotoxicity remains elusive. PMID:16540590
Recent Progress in Mouse Models for Tumor Suppressor Genes and its Implications in Human Cancer
Inoue, Kazushi; Fry, Elizabeth A.; Taneja, Pankaj
Gain-of-function mutations in oncogenes and loss-of-function mutations in tumor suppressor genes (TSG) lead to cancer. In most human cancers, these mutations occur in somatic tissues. However, hereditary forms of cancer exist for which individuals are heterozygous for a germline mutation in a TSG locus at birth. The second allele is frequently inactivated by gene deletion, point mutation, or promoter methylation in classical TSGs that meet Knudson�s two-hit hypothesis. Conversely, the second allele remains as wild-type, even in tumors in which the gene is haplo-insufficient for tumor suppression. This article highlights the importance of PTEN, APC, and other tumor suppressors for counteracting aberrant PI3K, ?-catenin, and other oncogenic signaling pathways. We discuss the use of gene-engineered mouse models (GEMM) of human cancer focusing on Pten and Apc knockout mice that recapitulate key genetic events involved in initiation and progression of human neoplasia. Finally, the therapeutic potential of targeting these tumor suppressor and oncogene signaling networks is discussed. PMID:23843721
Systems analysis of primary Sj�gren's syndrome pathogenesis in salivary glands identifies shared pathways in human and a mouse model
Introduction Primary Sj�gren's syndrome (pSS) is a chronic autoimmune disease with complex etiopathogenesis. Despite extensive studies to understand the disease process utilizing human and mouse models, the intersection between these species remains elusive. To address this gap, we utilized a novel systems biology approach to identify disease-related gene modules and signaling pathways that overlap between humans and mice. Methods Parotid gland tissues were harvested from 24 pSS and 16 non-pSS sicca patients and 25 controls. For mouse studies, salivary glands were harvested from C57BL/6.NOD-Aec1Aec2 mice at various times during development of pSS-like disease. RNA was analyzed with Affymetrix HG U133+2.0 arrays for human samples and with MOE430+2.0 arrays for mouse samples. The images were processed with Affymetrix software. Weighted-gene co-expression network analysis was used to identify disease-related and functional pathways. Results Nineteen co-expression modules were identified in human parotid tissue, of which four were significantly upregulated and three were downregulated in pSS patients compared with non-pSS sicca patients and controls. Notably, one of the human disease-related modules was highly preserved in the mouse model, and was enriched with genes involved in immune and inflammatory responses. Further comparison between these two species led to the identification of genes associated with leukocyte recruitment and germinal center formation. Conclusion Our systems biology analysis of genome-wide expression data from salivary gland tissue of pSS patients and from a pSS mouse model identified common dysregulated biological pathways and molecular targets underlying critical molecular alterations in pSS pathogenesis. PMID:23116360
Novel insights into the relationships between dendritic cell subsets in human and mouse revealed by genome-wide expression profiling
Robbins, Scott H; Walzer, Thierry; Demb�l�, Doulaye; Thibault, Christelle; Defays, Axel; Bessou, Gilles; Xu, Huichun; Vivier, Eric; Sellars, MacLean; Pierre, Philippe; Sharp, Franck R; Chan, Susan; Kastner, Philippe; Dalod, Marc
Background Dendritic cells (DCs) are a complex group of cells that play a critical role in vertebrate immunity. Lymph-node resident DCs (LN-DCs) are subdivided into conventional DC (cDC) subsets (CD11b and CD8? in mouse; BDCA1 and BDCA3 in human) and plasmacytoid DCs (pDCs). It is currently unclear if these various DC populations belong to a unique hematopoietic lineage and if the subsets identified in the mouse and human systems are evolutionary homologs. To gain novel insights into these questions, we sought conserved genetic signatures for LN-DCs and in vitro derived granulocyte-macrophage colony stimulating factor (GM-CSF) DCs through the analysis of a compendium of genome-wide expression profiles of mouse or human leukocytes. Results We show through clustering analysis that all LN-DC subsets form a distinct branch within the leukocyte family tree, and reveal a transcriptomal signature evolutionarily conserved in all LN-DC subsets. Moreover, we identify a large gene expression program shared between mouse and human pDCs, and smaller conserved profiles shared between mouse and human LN-cDC subsets. Importantly, most of these genes have not been previously associated with DC function and many have unknown functions. Finally, we use compendium analysis to re-evaluate the classification of interferon-producing killer DCs, lin-CD16+HLA-DR+ cells and in vitro derived GM-CSF DCs, and show that these cells are more closely linked to natural killer and myeloid cells, respectively. Conclusion Our study provides a unique database resource for future investigation of the evolutionarily conserved molecular pathways governing the ontogeny and functions of leukocyte subsets, especially DCs. PMID:18218067
Synergistic role of Spry2 inactivation and c-Met upregulation in mouse and human hepatocarcinogenesis
Lee, Susie A.; Ladu, Sara; Evert, Matthias; Dombrowski, Frank; De Murtas, Valentina; Chen, Xin; Calvisi, Diego F.
Sprouty2 (Spry2), a negative feedback regulator of the Ras/MAPK pathway, is frequently downregulated in human hepatocellular carcinoma (HCC). In the present investigation, we tested the hypothesis that loss of Spry2 cooperates with unconstrained activation of the c-Met protooncogene to induce hepatocarcinogenesis via in vitro and in vivo approaches. We found coordinated down-regulation of Spry2 protein expression and activation of c-Met as well as its downstream effectors ERK and AKT in a subset of human HCC samples with poor outcome. Mechanistic studies revealed that Spry2 function is disrupted in human HCC via multiple mechanisms at both transcriptional and post-transcriptional level, including promoter hypermethylation, loss of heterozygosity, and proteosomal degradation by neural precursor cell expressed, developmentally down-regulated 4 (NEDD4). In HCC cell lines, Spry2 overexpression inhibits c-Met-induced cell proliferation as well as ERK and AKT activation, whereas loss of Spry2 potentiates c-Met signaling. Most importantly, we show that blocking Spry2 activity via a dominant negative form of Spry2 cooperates with c-Met to promote hepatocarcinogenesis in the mouse liver by sustaining proliferation and angiogenesis. The tumors exhibited high levels of activated ERK and AKT, recapitulating the subgroup of human HCC with a clinically aggressive phenotype. Conclusion The occurrence of frequent genetic, epigenetic and biochemical events leading to Spry2 inactivation provides solid evidence that Spry2 functions as tumor suppressor gene in liver cancer. Coordinated deregulation of Spry2 and c-Met signaling may be a pivotal oncogenic mechanism responsible for unrestrained activation of ERK and AKT pathways in human hepatocarcinogenesis. PMID:20683950
In vitro metabolism of the analgesic bicifadine in the mouse, rat, monkey, and human.
Erickson, David A; Hollfelder, Stacy; Tenge, Justin; Gohdes, Mark; Burkhardt, Jeffrey J; Krieter, Philip A
The in vitro metabolism of [(14)C]bicifadine by hepatic microsomes and hepatocytes from mouse, rat, monkey, and human was compared using radiometric high-performance liquid chromatography and liquid chromatography/tandem mass spectrometry. Two main metabolic pathways were identified in all four species. One pathway was an NADPH-dependent pathway in which the methyl group was oxidized to form a hydroxymethyl metabolite (M2). Its formation was inhibited in human microsomes only by quinidine, a CYP2D6 inhibitor. In incubations with individual cDNA-expressed human cytochromes P450, M2 was formed only by CYP2D6 and CYP1A2, with CYP2D6 activity 6-fold greater than that of CYP1A2. M2 was oxidized further to the carboxylic acid metabolite (M3) by hepatocytes from all four species. The second major metabolic pathway was an NADPH-independent oxidation at the C2 position of the pyrrolidine ring, forming a lactam metabolite (M12). This reaction was almost completely inhibited in human hepatic microsomes and mitochondria by the monoamine oxidase (MAO)-B-specific inhibitor selegiline. Clorgyline, a specific inhibitor of MAO-A, was less effective in inhibiting M12 formation. Other metabolic pathways of variable significance among the four species included the formation of carbamoyl-O-glucuronide, hydroxymethyl lactam, and carboxyl lactam. Overall, the data indicate that the primary enzymes responsible for the primary metabolism of bicifadine in humans are MAO-B and CYP2D6. PMID:17881661
Systemic Disease-Induced Salivary Biomarker Profiles in Mouse Models of Melanoma and Non-Small Cell Lung Cancer
Gao, Kai; Zhou, Hui; Zhang, Lei; Lee, Jin Wook; Zhou, Qing; Hu, Shen; Wolinsky, Lawrence E.; Farrell, James; Eibl, Guido; Wong, David T.
Background Saliva (oral fluids) is an emerging biofluid poised for detection of clinical diseases. Although the rationale for oral diseases applications (e.g. oral cancer) is intuitive, the rationale and relationship between systemic diseases and saliva biomarkers are unclear. Methodology/Principal Findings In this study, we used mouse models of melanoma and non-small cell lung cancer and compared the transcriptome biomarker profiles of tumor-bearing mice to those of control mice. Microarray analysis showed that salivary transcriptomes were significantly altered in tumor-bearing mice vs. controls. Significant overlapping among transcriptomes of mouse tumors, serum, salivary glands and saliva suggests that salivary biomarkers have multiple origins. Furthermore, we identified that the expression of two groups of significantly altered transcription factors (TFs) Runx1, Mlxipl, Trim30 and Egr1, Tbx1, Nr1d1 in salivary gland tissue of melanoma-bearing mice can potentially be responsible for 82.6% of the up-regulated gene expression and 62.5% of the down-regulated gene expression, respectively, in the saliva of melanoma-bearing mice. We also showed that the ectopic production of nerve growth factor (NGF) in the melanoma tumor tissue as a tumor-released mediator can induce expression of the TF Egr-1 in the salivary gland. Conclusions Taken together, our data support the conclusion that upon systemic disease development, significant changes can occur in the salivary biomarker profile. Although the origins of the disease-induced salivary biomarkers may be both systemic and local, stimulation of salivary gland by mediators released from remote tumors plays an important role in regulating the salivary surrogate biomarker profiles. PMID:19517020
Biology of human skin transplanted to the nude mouse: I. Response to agents which modify epidermal proliferation.
Krueger, G G; Shelby, J
To accept human skin transplanted to the congenitally athymic (nude) mouse as a system to study human skin and its physiologic and pathologic states, it must be demonstrated that skin so maintained retains its function as a biologic unit. We have found that responses of grafted human skin and nude mouse skin to various agents differ. This difference in response has been utilized to assess barrier function and proliferative capacity of human skin grafts. Human skin grafts undergo a proliferative response when 10 ng of the tumor promoter, 12-O-tetradecanoyl phorbol 13-acetate (TPA) is applied. Nudes do not respond to this dose. Increasing the dose to 100 ng of TPA evokes a response in both. However, only in the human skin grafts can this response be blocked with betamethasone valerate (BV). In that human skin grafts do not take on their hosts' responsiveness, and the response of domestic pig skin to these agents before and after grafting is identical, the conclusion is reached that human skin appears to retain its inherent biologic unit function. The data also demonstrate some of the potential of this system to study kinetics of the epidermis of human skin. PMID:7017014
Experimental models of lymphoproliferative disease. The mouse as a model for human non-Hodgkin's lymphomas and related leukemias.
Pattengale, P. K.; Taylor, C. R.
The present review focuses on the mouse as an experimental immunopathologic model for human non-Hodgkin's lymphomas and related leukemias. Immunomorphologic evidence is presented that clearly demonstrates that B- and T-cell subtypes of mouse (murine) lymphoma/leukemia closely resemble and are analogous to B- and T-cell subtypes of human lymphoma/leukemia as defined by recently proposed immunomorphologic classifications. Further evidence is presented that favors the hypothesis that certain types of murine and human B-cell lymphoma develop out of prodromal, prelymphomatous states, which exhibit antecedent morphologic and immunologic abnormalities. The many experimental advantages of the murine systems are stressed, as well as the concept that the presently defined immunomorphologic approach should be effectively combined with molecular and cytogenetic parameters. Images Table 6 Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 10 Table 9 Figure 11 Figure 12 Figure 13 PMID:6605691
Mouse models of dominant ACTA1 disease recapitulate human disease and provide insight into therapies.
Ravenscroft, Gianina; Jackaman, Connie; Bringans, Scott; Papadimitriou, John M; Griffiths, Lisa M; McNamara, Elyshia; Bakker, Anthony J; Davies, Kay E; Laing, Nigel G; Nowak, Kristen J
Mutations in the skeletal muscle ?-actin gene (ACTA1) cause a range of pathologically defined congenital myopathies. Most patients have dominant mutations and experience severe skeletal muscle weakness, dying within one year of birth. To determine mutant ACTA1 pathobiology, transgenic mice expressing ACTA1(D286G) were created. These Tg(ACTA1)(D286G) mice were less active than wild-type individuals. Their skeletal muscles were significantly weaker by in vitro analyses and showed various pathological lesions reminiscent of human patients, however they had a normal lifespan. Mass spectrometry revealed skeletal muscles from Tg(ACTA1)(D286G) mice contained ?25% ACTA1(D286G) protein. Tg(ACTA1)(D286G) mice were crossed with hemizygous Acta1(+/-) knock-out mice to generate Tg(ACTA1)(D286G)(+/+).Acta1(+/-) offspring that were homozygous for the transgene and hemizygous for the endogenous skeletal muscle ?-actin gene. Akin to most human patients, skeletal muscles from these offspring contained approximately equal proportions of ACTA1(D286G) and wild-type actin. Strikingly, the majority of these mice presented with severe immobility between postnatal Days 8 and 17, requiring euthanasia. Their skeletal muscles contained extensive structural abnormalities as identified in severely affected human patients, including nemaline bodies, actin accumulations and widespread sarcomeric disarray. Therefore we have created valuable mouse models, one of mild dominant ACTA1 disease [Tg(ACTA1)(D286G)], and the other of severe disease, with a dramatically shortened lifespan [Tg(ACTA1)(D286G)(+/+).Acta1(+/-)]. The correlation between mutant ACTA1 protein load and disease severity parallels effects in ACTA1 families and suggests altering this ratio in patient muscle may be a therapy for patients with dominant ACTA1 disease. Furthermore, ringbinden fibres were observed in these mouse models. The presence of such features suggests that perhaps patients with ringbinden of unknown genetic origin should be considered for ACTA1 mutation screening. This is the first experimental, as opposed to observational, evidence that mutant protein load determines the severity of ACTA1 disease. PMID:21303860
Mouse and Human BAC Transgenes Recapitulate Tissue-Specific Expression of the Vitamin D Receptor in Mice and Rescue the VDR-Null Phenotype
Lee, Seong Min; Bishop, Kathleen A.; Goellner, Joseph J.; O'Brien, Charles A.
The biological actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are mediated by the vitamin D receptor (VDR), which is expressed in numerous target tissues in a cell type-selective manner. Recent studies using genomic analyses and recombineered bacterial artificial chromosomes (BACs) have defined the specific features of mouse and human VDR gene loci in vitro. In the current study, we introduced recombineered mouse and human VDR BACs as transgenes into mice and explored their expression capabilities in vivo. Individual transgenic mouse strains selectively expressed BAC-derived mouse or human VDR proteins in appropriate vitamin D target tissues, thereby recapitulating the tissue-specific expression of endogenous mouse VDR. The mouse VDR transgene was also regulated by 1,25(OH)2D3 and dibutyryl-cAMP. When crossed into a VDR-null mouse background, both transgenes restored wild-type basal as well as 1,25(OH)2D3-inducible gene expression patterns in the appropriate tissues. This maneuver resulted in the complete rescue of the aberrant phenotype noted in the VDR-null mouse, including systemic features associated with altered calcium and phosphorus homeostasis and disrupted production of parathyroid hormone and fibroblast growth factor 23, and abnormalities associated with the skeleton, kidney, parathyroid gland, and the skin. This study suggests that both mouse and human VDR transgenes are capable of recapitulating basal and regulated expression of the VDR in the appropriate mouse tissues and restore 1,25(OH)2D3 function. These results provide a baseline for further dissection of mechanisms integral to mouse and human VDR gene expression and offer the potential to explore the consequence of selective mutations in VDR proteins in vivo. PMID:24693968
Molecular Determinants of Severe Acute Respiratory Syndrome Coronavirus Pathogenesis and Virulence in Young and Aged Mouse Models of Human Disease
Yount, Boyd; Agnihothram, Sudhakar; Page, Carly; Donaldson, Eric; Roberts, Anjeanette; Vogel, Leatrice; Woodruff, Becky; Scorpio, Diana; Subbarao, Kanta; Baric, Ralph S.
SARS coronavirus (SARS-CoV) causes severe acute respiratory tract disease characterized by diffuse alveolar damage and hyaline membrane formation. This pathology often progresses to acute respiratory distress (such as acute respiratory distress syndrome [ARDS]) and atypical pneumonia in humans, with characteristic age-related mortality rates approaching 50% or more in immunosenescent populations. The molecular basis for the extreme virulence of SARS-CoV remains elusive. Since young and aged (1-year-old) mice do not develop severe clinical disease following infection with wild-type SARS-CoV, a mouse-adapted strain of SARS-CoV (called MA15) was developed and was shown to cause lethal infection in these animals. To understand the genetic contributions to the increased pathogenesis of MA15 in rodents, we used reverse genetics and evaluated the virulence of panels of derivative viruses encoding various combinations of mouse-adapted mutations. We found that mutations in the viral spike (S) glycoprotein and, to a much less rigorous extent, in the nsp9 nonstructural protein, were primarily associated with the acquisition of virulence in young animals. The mutations in S likely increase recognition of the mouse angiotensin-converting enzyme 2 (ACE2) receptor not only in MA15 but also in two additional, independently isolated mouse-adapted SARS-CoVs. In contrast to the findings for young animals, mutations to revert to the wild-type sequence in nsp9 and the S glycoprotein were not sufficient to significantly attenuate the virus compared to other combinations of mouse-adapted mutations in 12-month-old mice. This panel of SARS-CoVs provides novel reagents that we have used to further our understanding of differential, age-related pathogenic mechanisms in mouse models of human disease. PMID:22072787
Antibody therapy to human L1CAM in a transgenic mouse model blocks local tumor growth but induces EMT.
Doberstein, Kai; Harter, Patrick N; Haberkorn, Uwe; Bretz, Niko P; Arnold, Bernd; Carretero, Rafael; Moldenhauer, Gerhard; Mittelbronn, Michel; Altevogt, Peter
L1 cell adhesion molecule (L1CAM) is overexpressed in many human cancers, confers bad prognosis and augments cell motility, invasion and metastasis. Results from xenograft mouse models suggested that L1CAM antibodies might be promising tools for cancer therapy. Here, we generated human L1CAM-transgenic mice to study therapeutic efficacy and putative side effects in a model system. We established three transgenic lines (M2, M3 and F4) expressing the human L1CAM transgene in brain, kidney and colon with decreasing intensity (M2, M3 > F4). The expression pattern was similar to that of L1CAM in humans. No interference of the transgene with the expression of endogenous L1CAM was observed. Immunohistochemical analysis revealed correct expression of the transgene in mouse cortex and collective duct of the kidney. Injection of (125)I-labeled L1CAM antibodies resulted in specific enrichment in the kidney but not in the brain. The injection of the therapeutic anti-human L1CAM mAb L1-9.3/2a into transgenic mice even at high doses did not cause behavioral changes or other side effects. Similar results were obtained using a mouse specific L1CAM mAb in normal mice. Tumor therapy experiments were performed using syngeneic mouse tumor cells (RET melanoma and Panc02 pancreatic adenocarcinoma) transduced with human L1CAM. MAb L1-9.3/2a efficiently and specifically attenuated local tumor growth in both model systems without apparent side effects. The therapeutic effect was dependent on immune effector mechanisms. Analysis of Panc02-huL1CAM tumors after therapy showed elevated levels of EGF and evidence of immune-induced epithelial-mesenchymal transition. The results suggest that our transgenic mice are valuable tools to study L1CAM-based antibody therapy. PMID:25230579
Oxygen Sensing Mesenchymal Progenitors Promote Neo-Vasculogenesis in a Humanized Mouse Model In Vivo
Hofmann, Nicole A.; Ortner, Anna; Jacamo, Rodrigo O.; Reinisch, Andreas; Schallmoser, Katharina; Rohban, Rokhsareh; Etchart, Nathalie; Fruehwirth, Margareta; Beham-Schmid, Christine; Andreeff, Michael; Strunk, Dirk
Despite insights into the molecular pathways regulating hypoxia-induced gene expression, it is not known which cell types accomplish oxygen sensing during neo-vasculogenesis. We have developed a humanized mouse model of endothelial and mesenchymal progenitor co-transplantation to delineate the cellular compartments responsible for hypoxia response during vasculogenesis. Mesenchymal stem/progenitor cells (MSPCs) accumulated nuclear hypoxia-inducible transcription factor (HIF)-1? earlier and more sensitively than endothelial colony forming progenitor cells (ECFCs) in vitro and in vivo. Hypoxic ECFCs showed reduced function in vitro and underwent apoptosis within 24h in vivo when used without MSPCs. Surprisingly, only in MSPCs did pharmacologic or genetic inhibition of HIF-1? abrogate neo-vasculogenesis. HIF deletion in ECFCs caused no effect. ECFCs could be rescued from hypoxia-induced apoptosis by HIF-competent MSPCs resulting in the formation of patent perfused human vessels. Several angiogenic factors need to act in concert to partially substitute mesenchymal HIF-deficiency. Results demonstrate that ECFCs require HIF-competent vessel wall progenitors to initiate vasculogenesis in vivo and to bypass hypoxia-induced apoptosis. We describe a novel mechanistic role of MSPCs as oxygen sensors promoting vasculogenesis thus underscoring their importance for the development of advanced cellular therapies. PMID:22970226
The Gne M712T Mouse as a Model for Human Glomerulopathy
Kakani, Sravan; Yardeni, Tal; Poling, Justin; Ciccone, Carla; Niethamer, Terren; Klootwijk, Enriko D.; Manoli, Irini; Darvish, Daniel; Hoogstraten-Miller, Shelley; Zerfas, Patricia; Tian, E.; Ten Hagen, Kelly G.; Kopp, Jeffrey B.; Gahl, William A.; Huizing, Marjan
Pathological glomerular hyposialylation has been implicated in certain unexplained glomerulopathies, including minimal change nephrosis, membranous glomerulonephritis, and IgA nephropathy. We studied our previously established mouse model carrying a homozygous mutation in the key enzyme of sialic acid biosynthesis, N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. Mutant mice died before postnatal day 3 (P3) from severe glomerulopathy with podocyte effacement and segmental glomerular basement membrane splitting due to hyposialylation. Administration of the sialic acid precursor N-acetylmannosamine (ManNAc) led to improved sialylation and survival of mutant pups beyond P3. We determined the onset of the glomerulopathy in the embryonic stage. A lectin panel, distinguishing normally sialylated from hyposialylated glycans, used WGA, SNA, PNA, Jacalin, HPA, and VVA, indicating glomerular hyposialylation of predominantly O-linked glycoproteins in mutant mice. The glomerular glycoproteins nephrin and podocalyxin were hyposialylated in this unique murine model. ManNAc treatment appeared to ameliorate the hyposialylation status of mutant mice, indicated by a lectin histochemistry pattern similar to that of wild-type mice, with improved sialylation of both nephrin and podocalyxin, as well as reduced albuminuria compared with untreated mutant mice. These findings suggest application of our lectin panel for categorizing human kidney specimens based on glomerular sialylation status. Moreover, the partial restoration of glomerular architecture in ManNAc-treated mice highlights ManNAc as a potential treatment for humans affected with disorders of glomerular hyposialylation. PMID:22322304
Brucella ? 1,2 Cyclic Glucan Is an Activator of Human and Mouse Dendritic Cells
Martirosyan, Anna; P�rez-Gutierrez, Camino; Banchereau, Romain; Dutartre, H�l�ne; Lecine, Patrick; Dullaers, Melissa; Mello, Marielle; Pinto Salcedo, Suzana; Muller, Alexandre; Leserman, Lee; Levy, Yves; Zurawski, Gerard; Zurawski, Sandy; Moreno, Edgardo; Moriy�n, Ignacio; Klechevsky, Eynav; Banchereau, Jacques; Oh, SangKon; Gorvel, Jean-Pierre
Bacterial cyclic glucans are glucose polymers that concentrate within the periplasm of alpha-proteobacteria. These molecules are necessary to maintain the homeostasis of the cell envelope by contributing to the osmolarity of Gram negative bacteria. Here, we demonstrate that Brucella ? 1,2 cyclic glucans are potent activators of human and mouse dendritic cells. Dendritic cells activation by Brucella ? 1,2 cyclic glucans requires TLR4, MyD88 and TRIF, but not CD14. The Brucella cyclic glucans showed neither toxicity nor immunogenicity compared to LPS and triggered antigen-specific CD8+ T cell responses in vivo. These cyclic glucans also enhanced antigen-specific CD4+ and CD8+ T cell responses including cross-presentation by different human DC subsets. Brucella ? 1,2 cyclic glucans increased the memory CD4+ T cell responses of blood mononuclear cells exposed to recombinant fusion proteins composed of anti-CD40 antibody and antigens from both hepatitis C virus and Mycobacterium tuberculosis. Thus cyclic glucans represent a new class of adjuvants, which might contribute to the development of effective antimicrobial therapies. PMID:23166489
Functional characterization of a hybrid human-mouse interferon gamma receptor: evidence for species-specific interaction of the extracellular receptor domain with a putative signal transducer.
Hemmi, S; Merlin, G; Aguet, M
The human interferon gamma (IFN-gamma) receptor expressed in mouse cells displays binding properties indistinguishable from those of the resident receptor on human cells. Still, mouse cells expressing the human IFN-gamma receptor remain insensitive to human IFN-gamma. It is widely accepted that at least one species-specific cofactor encoded within human chromosome 21 is required for signal transduction. To define structural domains of the human IFN-gamma receptor responsible for this species-specific interaction, a hybrid between the human and the murine receptor was constructed and expressed in mouse L929 cells or in mouse L cell-derived SCC16-5 cells, which contain human chromosome 21. This hybrid receptor, which consisted of the extracellular domain of the human IFN-gamma receptor and the transmembrane and cytoplasmic domains of the murine IFN-gamma receptor, was found to bind human IFN-gamma with high affinity. However, only SCC16-5 cells expressing the human/mouse hybrid receptor were responsive to human IFN-gamma as revealed by enhanced expression of major histocompatibility complex class I antigens, induction of the transcription factor IRF-1, and induction of a partial antiviral state. These findings strongly suggest that IFN-gamma-mediated signal transduction requires a species-specific interaction of the extracellular portion of the known ligand-binding IFN-gamma receptor chain with an additional, presumably membrane-anchored receptor subunit. Images PMID:1532657
Translational Initiation at a Non-AUG Start Codon for Human and Mouse Negative Elongation Factor-B
Pan, Haihui; Zhao, Xiayan; Zhang, Xiaowen; Abouelsoud, Mohamed; Sun, Jianlong; April, Craig; Amleh, Asma; Fan, Jian-Bing; Hu, Yanfen; Li, Rong
Negative elongation factor (NELF), a four-subunit protein complex in metazoan, plays an important role in regulating promoter-proximal pausing of RNA polymerase II (RNAPII). Genetic studies demonstrate that the B subunit of mouse NELF (NELF-B) is critical for embryonic development and homeostasis in adult tissue. We report here that both human and mouse NELF-B proteins are translated from a non-AUG codon upstream of the annotated AUG. This non-AUG codon sequence is conserved in mammalian NELF-B but not NELF-B orthologs of lower metazoan. The full-length and a truncated NELF-B that starts at the first AUG codon both interact with the other three NELF subunits. Furthermore, these two forms of NELF-B have a similar impact on the transcriptomics and proliferation of mouse embryonic fibroblasts. These results strongly suggest that additional amino acid sequence upstream of the annotated AUG is dispensable for the essential NELF function in supporting cell growth in vitro. The majority of mouse adult tissues surveyed express the full-length NELF-B protein, and some contain a truncated NELF-B protein with the same apparent size as the AUG-initiated version. This result raises the distinct possibility that translational initiation of mouse NELF-B is regulated in a tissue-dependent manner. PMID:26010750
The genes encoding the glutamate receptor subunits KA1 and KA2 (GRIK4 and GRIK5) are located on separate chromosomes in human, mouse, and rat.
Szpirer, C; Moln�, M; Antonacci, R; Jenkins, N A; Finelli, P; Szpirer, J; Riviere, M; Rocchi, M; Gilbert, D J; Copeland, N G
The chromosomal localization of the human and rat genes encoding the kainate-preferring glutamate receptor subunits KA1 and KA2 (GRIK4 and GRIK5, respectively) was determined by Southern analysis of rat x mouse and human x mouse somatic cell hybrid panels and by fluorescence in situ hybridization. The localization of the mouse genes (Grik4 and Grik5) was established by interspecific backcross mapping. GRIK4 and GRIK5 are located on separate chromosomes (Chrs) in all species. GRIK4 mapped to human Chr 11q22.3, mouse Chr 9, and rat Chr 8. GRIK5 mapped to human Chr 19q13.2, mouse Chr 7, and rat Chr 1. The genes encoding the (R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-preferring subunit GluR4, or GluRD (GRIA4), the neural cell adhesion molecule (NCAM), the D2 dopamine receptor (DRD2), and the Thy-1 cell surface antigen (THY1) have all been previously mapped to the human Chr 11q22 region. The mapping of the human GRIK4 and GRIK5 genes confirms and extends the relationship between human Chr 11 and mouse Chr 9 and also human Chr 19 and mouse Chr 7. GRIK4 is the fifth gene shared by human Chr 11 and rat Chr 8, whereas GRIK5 is 1 out of the 12 genes that are located on both human Chr 19 and rat Chr 1. Our data extend the conserved synteny established between certain human, mouse, and rat Chrs. Images PMID:7527545
The human\\/mouse imprinted genes IGF2, H19, SNRPN and ZNF127 map to two conserved autosomal clusters in a marsupial
R. Toder; S. A. Wilcox; M. Smithwick; J. A. M. Graves
The four genesIGF2, H19, SNRPN andZNF127 are imprinted in mouse and human.IGF2 andH19 form one conserved cluster on the distal part of mouse chromosome 7 and human chromosome 11p15.5, whereasSNRPN andZNF127 form another on the middle of mouse chromosome 7 and on human chromosome 15q11-13. We have explored the evolution of these imprinted regions by cloning and mappingIGF2, H19, SNRPN
Complete Genome Sequences of One Human Respiratory Syncytial Antigenic Group A Virus from China and Its Four Mouse-Adapted Isolates
Zhang, Ke; He, Jie; Li, Cun; Bose, Michael E.; Henrickson, Kelly J.; Zhou, Jie
In this study, one human respiratory syncytial antigenic group A virus (HRSV-A-GZ08-0) and its four BALB/c mouse-adapted isolates were sequenced and elucidated. Nineteen nucleotides were mutated between HRSV-A-GZ08-0 and the four mouse-adapted isolates. PMID:25744999
Calculated and TLD-based absorbed dose estimates for I-131-labeled 3F8 monoclonal antibody in a human neuroblastoma xenograft nude mouse model
Omer Ugur; Andrew M. Scott; Lale Kostakoglu; T. Edmond Hui; Mary E. Masterson; Robert Febo; George Sgouros; Eddie Rosa; Bipin M. Mehta; Darrell R. Fisher; Nai-Kong V. Cheung; Steven M. Larson
Preclinical evaluation of the therapeutic potential of radiolabeled antibodies is commonly performed in a xenografted nude mouse model. To assess therapeutic efficacy it is important to estimate the absorbed dose to the tumor and normal tissues of the nude mouse. The current study was designed to accurately measure radiation does to human neuroblastoma xenografts and normal organs in nude mice
The Regulation of Nitric Oxide Synthase Isoform Expression in Mouse and Human Fallopian Tubes: Potential Insights for Ectopic Pregnancy
Hu, Junting; Ma, Shulan; Zou, Sien; Li, Xin; Cui, Peng; Weijdeg�rd, Birgitta; Wu, Gencheng; Shao, Ruijin; Billig, H�kan; Feng, Yi
Nitric oxide (NO) is highly unstable and has a half-life of seconds in buffer solutions. It is synthesized by NO-synthase (NOS), which has been found to exist in the following three isoforms: neuro nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS). NOS activity is localized in the reproductive tracts of many species, although direct evidence for NOS isoforms in the Fallopian tubes of mice is still lacking. In the present study, we investigated the expression and regulation of NOS isoforms in the mouse and human Fallopian tubes during the estrous and menstrual cycles, respectively. We also measured isoform expression in humans with ectopic pregnancy and in mice treated with lipopolysaccharide (LPS). Our results confirmed the presence of different NOS isoforms in the mouse and human Fallopian tubes during different stages of the estrous and menstrual cycles and showed that iNOS expression increased in the Fallopian tubes of women with ectopic pregnancy and in LPS-treated mice. Elevated iNOS activity might influence ovulation, cilia beats, contractility, and embryo transportation in such a manner as to increase the risk of ectopic pregnancy. This study has provided morphological and molecular evidence that NOS isoforms are present and active in the human and mouse Fallopian tubes and suggests that iNOS might play an important role in both the reproductive cycle and infection-induced ectopic pregnancies. PMID:25546387
Uranyl nitrate inhibits lactate gluconeogenesis in isolated human and mouse renal proximal tubules: A {sup 13}C-NMR study
Renault, Sophie; Faiz, Hassan; Gadet, Rudy; Ferrier, Bernard; Martin, Guy; Baverel, Gabriel [Metabolomique et Maladies Metaboliques, Institut National de la Sante et de la recherche Medicale, Unit 820, Faculte de Medecine R.T.H. Laennec, Universite de Lyon, 7-11 rue G. Paradin, 69372 Lyon Cedex 08 (France); Conjard-Duplany, Agnes, E-mail: firstname.lastname@example.com [Metabolomique et Maladies Metaboliques, Institut National de la Sante et de la recherche Medicale, Unit 820, Faculte de Medecine R.T.H. Laennec, Universite de Lyon, 7-11 rue G. Paradin, 69372 Lyon Cedex 08 (France)
As part of a study on uranium nephrotoxicity, we investigated the effect of uranyl nitrate in isolated human and mouse kidney cortex tubules metabolizing the physiological substrate lactate. In the millimolar range, uranyl nitrate reduced lactate removal and gluconeogenesis and the cellular ATP level in a dose-dependent fashion. After incubation in phosphate-free Krebs-Henseleit medium with 5 mM L-[1-{sup 13}C]-, or L-[2-{sup 13}C]-, or L-[3-{sup 13}C]lactate, substrate utilization and product formation were measured by enzymatic and NMR spectroscopic methods. In the presence of 3 mM uranyl nitrate, glucose production and the intracellular ATP content were significantly reduced in both human and mouse tubules. Combination of enzymatic and NMR measurements with a mathematical model of lactate metabolism revealed an inhibition of fluxes through lactate dehydrogenase and the gluconeogenic enzymes in the presence of 3 mM uranyl nitrate; in human and mouse tubules, fluxes were lowered by 20% and 14% (lactate dehydrogenase), 27% and 32% (pyruvate carboxylase), 35% and 36% (phosphoenolpyruvate carboxykinase), and 39% and 45% (glucose-6-phosphatase), respectively. These results indicate that natural uranium is an inhibitor of renal lactate gluconeogenesis in both humans and mice.
microPIR2: a comprehensive database for human�mouse comparative study of microRNA�promoter interactions
Piriyapongsa, Jittima; Bootchai, Chaiwat; Ngamphiw, Chumpol; Tongsima, Sissades
microRNA (miRNA)�promoter interaction resource (microPIR) is a public database containing over 15 million predicted miRNA target sites located within human promoter sequences. These predicted targets are presented along with their related genomic and experimental data, making the microPIR database the most comprehensive repository of miRNA promoter target sites. Here, we describe major updates of the microPIR database including new target predictions in the mouse genome and revised human target predictions. The updated database (microPIR2) now provides ?80 million human and 40 million mouse predicted target sites. In addition to being a reference database, microPIR2 is a tool for comparative analysis of target sites on the promoters of human�mouse orthologous genes. In particular, this new feature was designed to identify potential miRNA�promoter interactions conserved between species that could be stronger candidates for further experimental validation. We also incorporated additional supporting information to microPIR2 such as nuclear and cytoplasmic localization of miRNAs and miRNA�disease association. Extra search features were also implemented to enable various investigations of targets of interest. Database URL: http://www4a.biotec.or.th/micropir2 PMID:25425035
Mouse Forward Genetics in the Study of the Peripheral Nervous System and Human Peripheral Neuropathy
Darlene S. Douglas; Brian Popko
Forward genetics, the phenotype-driven approach to investigating gene identity and function, has a long history in mouse genetics.\\u000a Random mutations in the mouse transcend bias about gene function and provide avenues towards unique discoveries. The study\\u000a of the peripheral nervous system is no exception; from historical strains such as the trembler mouse, which led to the identification of PMP22 as
Genetic mapping in human and mouse of the locus encoding TRBP, a protein that binds the TAR region of the human immunodeficiency virus (HIV-1)
Kozak, C.A.; Gatignol, A.; Graham, K. [National Inst. of Allergy and Infectious Diseases, Behesda, MD (United States)] [and others] [National Inst. of Allergy and Infectious Diseases, Behesda, MD (United States); and others
Productive infection with HIV-1, the virus responsible for AIDS, requires the involvement of host cell factors for completion of the replicative cycle, but the identification of these factors and elucidation of their specific functions has been difficult. A human cDNA, TRBP, was recently cloned and characterized as a positive regulator of gene expression that binds to the TAR region of the HIV-1 genome. Here we demonstrate that this factor is encoded by a gene, TARBP2, that maps to human chromosome 12 and mouse chromosome 15, and we also identify and map one human pseudogene (TARBP2P) and two mouse TRBP-related sequences. The map location of the expressed gene identifies it as a candidate for the previously identified factor encoded on human chromosome 12 that has been shown to be important for expression of HIV-1 genes. Western blotting indicates that despite high sequence conservation in human and mouse, the TARBP2 protein differs in apparent size in primate and rodent cells. 41 refs., 5 figs., 1 tab.
Identification of genome-derived vaccine candidates conserved between human and mouse-adapted strains of H. pylori.
Moise, Leonard; McMurry, Julie A; Pappo, Jacques; Lee, Dong-Soo; Moss, Steven F; Martin, William D; De Groot, Anne S
Computational methods accelerate vaccine development by rapid identification of potential vaccine candidates. We screened the Helicobacter pylori J99 and 26695 genomes for T-cell epitopes using the epitope mapping algorithm EpiMatrix and selected 150 sequences for experimental validation in a pre-clinical mouse model. Because strains of H. pylori that infect humans do not generally infect mice, and the sequence of the mouse-adapted "Sydney" strain (SS1) is not publicly available, we used targeted PCR to confirm that the epitopes we computationally predicted from the human H. pylori isolates J99 and 26695 are conserved in SS1. Epitopes conserved in SS1 were further analyzed for binding to MHC in vitro and for antigenicity in infected mice to select candidates for an epitope-based vaccine. PMID:18376134
Chromosomal mapping of the human and mouse homologues of two new members of the AP-2 family of transcription factors
Williamson, J.A.; Sheer, D. [ICRF, London (United Kingdom)] [ICRF, London (United Kingdom); Bosher, J.M. [Hammersmith Hospital, Longon (United Kingdom)] [and others] [Hammersmith Hospital, Longon (United Kingdom); and others
The AP-2 transcription factor has been shown to play an important role in the development of tissues of ectodermal origin and has also been implicated in mammary oncogenesis. It has recently been found that AP-2 is encoded by a family of related genes, AP-2{alpha}, AP-2{beta}, and AP-2{gamma}. As a further step in understanding the role of each of these genes has in development, we have used fluorescence in situ hybridization to map the chromosomal locations of the mouse and human homologues of the newly isolated AP-2{beta} and AP-2{gamma} genes. Tcfap2b and Tcfap2c map to mouse chromosomes 1A2-4 and 2H3-4, respectively, while TFAP2B and TFAP2C map to human chromosomes 6p12 and 20q13.2, the later being a region that is frequently amplified in breast carcinoma. 20 refs., 1 fig., 1 tab.
Feline head and neck squamous cell carcinoma: a natural model for the human disease and development of a mouse model.
Tannehill-Gregg, S H; Levine, A L; Rosol, T J
Head and neck squamous cell carcinoma (H/N SCC) is a devastating disease in humans and cats, and shares similar features between the two species. The large population of pet cats in the United States, along with the high incidence of oral SCC in the cat, makes the cat an attractive candidate as a natural model for the human disease. There are similarities in pathology, progression, outcome, resistance to treatment, possible aetiologies and p53 expression, and we discuss the benefits of the cat as a natural model. We describe the development of a nude mouse xenograft model of feline oral SCC using the SCCF1 cell line transfected with a luciferase expression construct. In vivo tumour growth and metastasis were measured using serial bioluminescent imaging, and tumours grew best in the subcutis. The cat and nude mouse models will be useful to investigate the pathogenesis and the molecular basis of H/N SCC, and for preclinical drug screening. PMID:19754818
Targeting of MCL-1 kills MYC-driven mouse and human lymphomas even when they bear mutations in p53
Kelly, Gemma L.; Grabow, Stephanie; Glaser, Stefan P.; Fitzsimmons, Leah; Aubrey, Brandon J.; Okamoto, Toru; Valente, Liz J.; Robati, Mikara; Tai, Lin; Fairlie, W. Douglas; Lee, Erinna F.; Lindstrom, Mikael S.; Wiman, Klas G.; Huang, David C.S.; Bouillet, Philippe; Rowe, Martin; Rickinson, Alan B.; Herold, Marco J.; Strasser, Andreas
The transcriptional regulator c-MYC is abnormally overexpressed in many human cancers. Evasion from apoptosis is critical for cancer development, particularly c-MYC-driven cancers. We explored which anti-apoptotic BCL-2 family member (expressed under endogenous regulation) is essential to sustain c-MYC-driven lymphoma growth to reveal which should be targeted for cancer therapy. Remarkably, inducible Cre-mediated deletion of even a single Mcl-1 allele substantially impaired the growth of c-MYC-driven mouse lymphomas. Mutations in p53 could diminish but not obviate the dependency of c-MYC-driven mouse lymphomas on MCL-1. Importantly, targeting of MCL-1 killed c-MYC-driven human Burkitt lymphoma cells, even those bearing mutations in p53. Given that loss of one allele of Mcl-1 is well tolerated in healthy tissues, our results suggest that therapeutic targeting of MCL-1 would be an attractive therapeutic strategy for MYC-driven cancers. PMID:24395247
Biostability of Batracylin: Incubation of batracylin in mouse and human plasma for as long as 48 h did not produce significant degradation
Batracyclin Pharmacology Abstract Division of Cancer Treatment and Diagnosis National Cancer Institute Biostability of Batracylin: Incubation of batracylin in mouse and human plasma for as long as 48 h did not produce significant degradation.
Transmission Properties of Human PrP 102L Prions Challenge the Relevance of Mouse Models of GSS.
Asante, Emmanuel A; Grimshaw, Andrew; Smidak, Michelle; Jakubcova, Tatiana; Tomlinson, Andrew; Jeelani, Asif; Hamdan, Shyma; Powell, Caroline; Joiner, Susan; Linehan, Jacqueline M; Brandner, Sebastian; Wadsworth, Jonathan D F; Collinge, John
Inherited prion disease (IPD) is caused by autosomal-dominant pathogenic mutations in the human prion protein (PrP) gene (PRNP). A proline to leucine substitution at PrP residue 102 (P102L) is classically associated with Gerstmann-Str�ussler-Scheinker (GSS) disease but shows marked clinical and neuropathological variability within kindreds that may be caused by variable propagation of distinct prion strains generated from either PrP 102L or wild type PrP. To-date the transmission properties of prions propagated in P102L patients remain ill-defined. Multiple mouse models of GSS have focused on mutating the corresponding residue of murine PrP (P101L), however murine PrP 101L, a novel PrP primary structure, may not have the repertoire of pathogenic prion conformations necessary to accurately model the human disease. Here we describe the transmission properties of prions generated in human PrP 102L expressing transgenic mice that were generated after primary challenge with ex vivo human GSS P102L or classical CJD prions. We show that distinct strains of prions were generated in these mice dependent upon source of the inoculum (either GSS P102L or CJD brain) and have designated these GSS-102L and CJD-102L prions, respectively. GSS-102L prions have transmission properties distinct from all prion strains seen in sporadic and acquired human prion disease. Significantly, GSS-102L prions appear incapable of transmitting disease to conventional mice expressing wild type mouse PrP, which contrasts strikingly with the reported transmission properties of prions generated in GSS P102L-challenged mice expressing mouse PrP 101L. We conclude that future transgenic modeling of IPDs should focus exclusively on expression of mutant human PrP, as other approaches may generate novel experimental prion strains that are unrelated to human disease. PMID:26135918
Transmission Properties of Human PrP 102L Prions Challenge the Relevance of Mouse Models of GSS
Asante, Emmanuel A.; Grimshaw, Andrew; Smidak, Michelle; Jakubcova, Tatiana; Tomlinson, Andrew; Jeelani, Asif; Hamdan, Shyma; Powell, Caroline; Joiner, Susan; Linehan, Jacqueline M.; Brandner, Sebastian; Wadsworth, Jonathan D. F.; Collinge, John
Inherited prion disease (IPD) is caused by autosomal-dominant pathogenic mutations in the human prion protein (PrP) gene (PRNP). A proline to leucine substitution at PrP residue 102 (P102L) is classically associated with Gerstmann-Str�ussler-Scheinker (GSS) disease but shows marked clinical and neuropathological variability within kindreds that may be caused by variable propagation of distinct prion strains generated from either PrP 102L or wild type PrP. To-date the transmission properties of prions propagated in P102L patients remain ill-defined. Multiple mouse models of GSS have focused on mutating the corresponding residue of murine PrP (P101L), however murine PrP 101L, a novel PrP primary structure, may not have the repertoire of pathogenic prion conformations necessary to accurately model the human disease. Here we describe the transmission properties of prions generated in human PrP 102L expressing transgenic mice that were generated after primary challenge with ex vivo human GSS P102L or classical CJD prions. We show that distinct strains of prions were generated in these mice dependent upon source of the inoculum (either GSS P102L or CJD brain) and have designated these GSS-102L and CJD-102L prions, respectively. GSS-102L prions have transmission properties distinct from all prion strains seen in sporadic and acquired human prion disease. Significantly, GSS-102L prions appear incapable of transmitting disease to conventional mice expressing wild type mouse PrP, which contrasts strikingly with the reported transmission properties of prions generated in GSS P102L-challenged mice expressing mouse PrP 101L. We conclude that future transgenic modeling of IPDs should focus exclusively on expression of mutant human PrP, as other approaches may generate novel experimental prion strains that are unrelated to human disease. PMID:26135918
Hepatitis B Virus Infection and Immunopathogenesis in a Humanized Mouse Model: Induction of Human-Specific Liver Fibrosis and M2-Like Macrophages
Bility, Moses T.; Cheng, Liang; Zhang, Zheng; Luan, Yan; Li, Feng; Chi, Liqun; Zhang, Liguo; Tu, Zhengkun; Gao, Yanhang; Fu, Yangxin; Niu, Junqi; Wang, Fusheng; Su, Lishan
The mechanisms of chronic HBV infection and immunopathogenesis are poorly understood due to a lack of a robust small animal model. Here we report the development of a humanized mouse model with both human immune system and human liver cells by reconstituting the immunodeficient A2/NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice with human HLA-A2 transgene) with human hematopoietic stem cells and liver progenitor cells (A2/NSG-hu HSC/Hep mice). The A2/NSG-hu HSC/Hep mouse supported HBV infection and approximately 75% of HBV infected mice established persistent infection for at least 4 months. We detected human immune responses, albeit impaired in the liver, chronic liver inflammation and liver fibrosis in infected animals. An HBV neutralizing antibody efficiently inhibited HBV infection and associated liver diseases in humanized mice. In addition, we found that the HBV mediated liver disease was associated with high level of infiltrated human macrophages with M2-like activation phenotype. Importantly, similar M2-like macrophage accumulation was confirmed in chronic hepatitis B patients with liver diseases. Furthermore, gene expression analysis showed that induction of M2-like macrophage in the liver is associated with accelerated liver fibrosis and necrosis in patients with acute HBV-induced liver failure. Lastly, we demonstrate that HBV promotes M2-like activation in both M1 and M2 macrophages in cell culture studies. Our study demonstrates that the A2/NSG-hu HSC/Hep mouse model is valuable in studying HBV infection, human immune responses and associated liver diseases. Furthermore, results from this study suggest a critical role for macrophage polarization in hepatitis B virus-induced immune impairment and liver pathology. PMID:24651854
Hepatitis B virus infection and immunopathogenesis in a humanized mouse model: induction of human-specific liver fibrosis and M2-like macrophages.
Bility, Moses T; Cheng, Liang; Zhang, Zheng; Luan, Yan; Li, Feng; Chi, Liqun; Zhang, Liguo; Tu, Zhengkun; Gao, Yanhang; Fu, Yangxin; Niu, Junqi; Wang, Fusheng; Su, Lishan
The mechanisms of chronic HBV infection and immunopathogenesis are poorly understood due to a lack of a robust small animal model. Here we report the development of a humanized mouse model with both human immune system and human liver cells by reconstituting the immunodeficient A2/NSG (NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ mice with human HLA-A2 transgene) with human hematopoietic stem cells and liver progenitor cells (A2/NSG-hu HSC/Hep mice). The A2/NSG-hu HSC/Hep mouse supported HBV infection and approximately 75% of HBV infected mice established persistent infection for at least 4 months. We detected human immune responses, albeit impaired in the liver, chronic liver inflammation and liver fibrosis in infected animals. An HBV neutralizing antibody efficiently inhibited HBV infection and associated liver diseases in humanized mice. In addition, we found that the HBV mediated liver disease was associated with high level of infiltrated human macrophages with M2-like activation phenotype. Importantly, similar M2-like macrophage accumulation was confirmed in chronic hepatitis B patients with liver diseases. Furthermore, gene expression analysis showed that induction of M2-like macrophage in the liver is associated with accelerated liver fibrosis and necrosis in patients with acute HBV-induced liver failure. Lastly, we demonstrate that HBV promotes M2-like activation in both M1 and M2 macrophages in cell culture studies. Our study demonstrates that the A2/NSG-hu HSC/Hep mouse model is valuable in studying HBV infection, human immune responses and associated liver diseases. Furthermore, results from this study suggest a critical role for macrophage polarization in hepatitis B virus-induced immune impairment and liver pathology. PMID:24651854
Feeder-dependent and feeder-independent iPS cell derivation from human and mouse adipose stem cells
Shigeki Sugii; Yasuyuki Kida; W Travis Berggren; Ronald M Evans
Adipose tissue is an abundantly available source of proliferative and multipotent mesenchymal stem cells with promising potential for regenerative therapeutics. We previously demonstrated that both human and mouse adipose-derived stem cells (ASCs) can be reprogrammed into induced pluripotent stem cells (iPSCs) with efficiencies higher than those that have been reported for other cell types. The ASC-derived iPSCs can be generated
Human Neural Stem Cells Genetically Modified to Overexpress Akt1 Provide Neuroprotection and Functional Improvement in Mouse Stroke Model
Hong J. Lee; Mi K. Kim; Hee J. Kim; Seung U. Kim; Rafael Linden
In a previous study, we have shown that human neural stem cells (hNSCs) transplanted in brain of mouse intracerebral hemorrhage (ICH) stroke model selectively migrate to the ICH lesion and induce behavioral recovery. However, low survival rate of grafted hNSCs in the brain precludes long-term therapeutic effect. We hypothesized that hNSCs overexpressing Akt1 transplanted into the lesion site could provide
The amino acid sequences of the carboxyl termini of human and mouse hepatic lipase influence cell surface association
Robert J. Brown; Joshua R. Schultz; Kerry W. S. Ko; John S. Hill; Tanya A. Ramsamy; Ann L. White; Daniel L. Sparks; Zemin Yao
Human hepatic lipase (hHL) mainly exists cell sur- face bound, whereas mouse HL (mHL) circulates in the blood stream. Studies have suggested that the carboxyl terminus of HL mediates cell surface binding. We prepared recombinant hHL, mHL, and chimeric proteins (hHLmt and mHLht) in which the carboxyl terminal 70 amino acids of hHL were ex- changed with the corresponding sequence
Clustering of six human 11p15 gene homologs within a 500-kb interval of proximal mouse chromosome 7
L. Stubbs; E. M. Rinchik; D. Johnson
Homologs of genes mapping to human chromosome 11p15 are located in three distinct, widely separated regions of mouse chromosome 7 (Mmu7). To date, six genes have been localized to the most proximal HSA11p15\\/Mmu7 homology region, including Ldh3 (encoding lactate dehydrogenase C), Ldh1 (lactate dehydrogenase A), Myod1 (myogenic differentiation factor-1), Tph (tryptophan hydroxlase), Saa1 (serum amyloid-A-1), and Kcnc1 (encoding a Shaw-type
High-resolution large-scale mosaic imaging using multiphoton microscopy to characterize transgenic mouse models of human neurological disorders
Diana L. Price; Sunny K. Chow; Natalie A. B. MacLean; Hiroyuki Hakozaki; Steve Peltier; Maryann E. Martone; Mark H. Ellisman
The thorough characterization of transgenic mouse models of human central nervous system diseases is a necessary step in realizing\\u000a the full benefit of using animal models to investigate disease processes and potential therapeutics. Because of the labor-\\u000a and resource-intensive nature of high-resolution imaging, detailed investigation of possible structural or biochemical alterations\\u000a in brain sections has typically focused on specific regions
Relative suppression of the sodium-dependent Vitamin C transport in mouse versus human lens epithelial cells.
Obrenovich, Mark E; Fan, Xingjun; Satake, Makoto; Jarvis, Simon M; Reneker, Lixing; Reddan, John R; Monnier, Vincent M
Vitamin C is a major antioxidant and UV absorbent in the human lens. In the rodent lens, the levels are very low for unknown reasons. Searching for clues to explain this suppression, we investigated the comparative uptake of Vitamin C in cultured human and mouse lens epithelial cells. When compared to human HLE-B3 lens epithelial cells, (14)C-ASA uptake was 4- to 10-fold impaired in confluent mouse lens 17EM15 (p < 0.0001) and 21EM15 (p < 0.001) cells, respectively. High glucose concentrations reduced the uptake by 30-50% in all cells (p < 0.005). Incubation of cells with 6-deoxy-6-fluoro-ascorbic (F-ASA), i.e. a probe specific for the sodium-dependent Vitamin C uptake (SVCT2), revealed a 10-fold uptake suppression into mouse 17EM15 relative to human HLE-B3 and JAR choriocarcinoma cells (a control), that could be overcome by overexpressing hSVCT2 using two different promoter constructs. The relative Vitamin C uptake differences suggest either low expression of SVCT2, molecular differences between the transporters themselves or their biological regulation, since a recent study has shown that exogenous feeding of ascorbic acid to rats increased only modestly lenticular uptake (Mody et al., Acta Ophthalmol Scand 83: 228-223, 2005). Elucidation of the mechanism by which SCVT2 activity is suppressed in mouse lens may help unravel a major question of evolutionary significance for night vision in nocturnal animals. PMID:16933033
Human Mesenchymal Stem Cells Prolong Survival and Ameliorate Motor Deficit through Trophic Support in Huntington's Disease Mouse Models
Yuan-Ta Lin; Yijuang Chern; Che-Kun James Shen; Hsin-Lan Wen; Ya-Chin Chang; Hung Li; Tzu-Hao Cheng; Hsiu Mei Hsieh-Li
We investigated the therapeutic potential of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) in Huntington's disease (HD) mouse models. Ten weeks after intrastriatal injection of quinolinic acid (QA), mice that received hBM-MSC transplantation showed a significant reduction in motor function impairment and increased survival rate. Transplanted hBM-MSCs were capable of survival, and inducing neural proliferation and differentiation in the QA-lesioned
Presence of mouse mammary tumour-like virus gene sequences may be associated with morphology of specific human breast cancer
J S Lawson; D D Tran; E Carpenter; C E Ford; W D Rawlinson; N J Whitaker; W Delprado
Background: Mouse mammary tumour virus (MMTV) has a proven role in breast carcinogenesis in wild mice and genetically susceptible in-bred mice. MMTV-like env gene sequences, which indicate the presence of a replication-competent MMTV-like virus, have been identified in some human breast cancers, but rarely in normal breast tissues. However, no evidence for a causal role of an MMTV-like virus in
Metabolism of propafenone and verapamil by cryopreserved human, rat, mouse and dog hepatocytes: comparison with metabolism in vivo
B. Reder-Hilz; M. Ullrich; M. Ringel; N. Hewitt; D. Utesch; F. Oesch; J. G. Hengstler
In the present study we examined the metabolism of [14C]propafenone (P) and [14C]verapamil (V) using cryopreserved human, dog (Beagle), rat (Sprague-Dawley) and mouse (NMRI) hepatocytes. The percentage ratios of the metabolites were identified after extraction by HPLC with UV and radioactivity detection. Phase-II metabolites were cleaved using ?-glucuronidase. Metabolism of the drugs by cryopreserved hepatocytes was compared with that in
In vivo sequestration of Plasmodium falciparum-infected human erythrocytes: a severe combined immunodeficiency mouse model for cerebral malaria
Cerebral malaria is a fatal complication of infection by Plasmodium falciparum in man. The neurological symptoms that characterize this form of malarial disease are accompanied by the adhesion of infected erythrocytes to the vasculature of the brain. To study this phenomenon in vivo, an acute phase severe combined immunodeficiency (SCID) mouse model was developed in which sequestration of P. falciparum-infected human erythrocytes took place. During acute cerebral malaria in humans, the expression of intercellular adhesion molecule-1 (ICAM-1) is induced in vascular endothelium by inflammatory reactions. Acute phase ICAM-1 expression can also be obtained in SCID mice. The endothelium of the midbrain region was the most responsive to such inflammatory stimulus. It is noteworthy that the reticular formation in the midbrain controls the level of consciousness, and loss of consciousness is a symptom of cerebral malaria. We found that infected human erythrocytes were retained 24 times more than normal erythrocytes in ICAM-1-positive mouse brain. Sequestration to the brain was reduced by anti-ICAM-1 antibodies. These in vivo results were confirmed by the binding of P. falciparum-infected erythrocytes to the ICAM-1-positive endothelium in tissue sections of mouse brain. We conclude that the SCID mouse serves as a versatile in vivo model that allows the study of P. falciparum- infected erythrocyte adhesion as it occurs in human cerebral malaria. Upregulation of ICAM-1 expression in the region of the midbrain correlates with increased retention of malaria-infected erythrocytes and with the symptoms of cerebral malaria. PMID:7650476
Human Atopic Dermatitis Skin-derived T Cells can Induce a Reaction in Mouse Keratinocytes in�vivo.
Martel, B C; Blom, L; Dyring-Andersen, B; Skov, L; Thestrup-Pedersen, K; Skov, S; Skak, K; Poulsen, L K
In atopic dermatitis (AD), the inflammatory response between skin-infiltrating T cells and keratinocytes is fundamental to the development of chronic lesional eczema. The aim of this study was to investigate whether skin-derived T cells from AD patients could induce an inflammatory response in mice through keratinocyte activation and consequently cause the development of eczematous lesions. Punch biopsies of the lesional skin from AD patients were used to establish skin-derived T cell cultures, which were transferred to NOD.Cg-Prkd(scid) Il2rg(tm1Sug) /JicTac (NOG) mice. We found that the subcutaneous injection of the human AD skin-derived T cells resulted in the migration of the human T cells from subcutis to the papillary dermis followed by the development of erythema and oedema in the mouse skin. Furthermore, the human T cells induced a transient proliferative response in the mouse keratinocytes shown as increased numbers of Ki-67(+) keratinocytes and increased epidermal thickness. Out of six established AD skin-derived T cell cultures, two were superior at inducing a skin reaction in the mice, and these cultures were found to contain >10% CCR10(+) T cells compared to <2% for the other cultures. In comparison, blood-derived in�vitro-differentiated Th2 cells only induced a weak response in a few of the mice. Thus, we conclude that human AD skin-derived T cells can induce a reaction in the mouse skin through the induction of a proliferative response in the mouse keratinocytes. PMID:25998164
Fibulin-4 and fibulin-5 in elastogenesis and beyond: Insights from mouse and human studies.
Papke, Christina L; Yanagisawa, Hiromi
The fibulin family of extracellular matrix/matricellular proteins is composed of long fibulins (fibulin-1, -2, -6) and short fibulins (fibulin-3, -4, -5, -7) and is involved in protein-protein interaction with the components of basement membrane and extracellular matrix proteins. Fibulin-1, -2, -3, -4, and -5 bind the monomeric form of elastin (tropoelastin) in vitro and fibulin-2, -3, -4, and -5 are shown to be involved in various aspects of elastic fiber development in vivo. In particular, fibulin-4 and -5 are critical molecules for elastic fiber assembly and play a non-redundant role during elastic fiber formation. Despite manifestation of systemic elastic fiber defects in all elastogenic tissues, fibulin-5 null (Fbln5(-/-)) mice have a normal lifespan. In contrast, fibulin-4 null (Fbln4(-/-)) mice die during the perinatal period due to rupture of aortic aneurysms, indicating differential functions of fibulin-4 and fibulin-5 in normal development. In this review, we will update biochemical characterization of fibulin-4 and fibulin-5 and discuss their roles in elastogenesis and outside of elastogenesis based on knowledge obtained from loss-of-function studies in mouse and in human patients with FBLN4 or FBLN5 mutations. Finally, we will evaluate therapeutic options for matrix-related diseases. PMID:24613575
Tissue-Specific Evolution of Protein Coding Genes in Human and Mouse
Kryuchkova-Mostacci, Nadezda; Robinson-Rechavi, Marc
Protein-coding genes evolve at different rates, and the influence of different parameters, from gene size to expression level, has been extensively studied. While in yeast gene expression level is the major causal factor of gene evolutionary rate, the situation is more complex in animals. Here we investigate these relations further, especially taking in account gene expression in different organs as well as indirect correlations between parameters. We used RNA-seq data from two large datasets, covering 22 mouse tissues and 27 human tissues. Over all tissues, evolutionary rate only correlates weakly with levels and breadth of expression. The strongest explanatory factors of purifying selection are GC content, expression in many developmental stages, and expression in brain tissues. While the main component of evolutionary rate is purifying selection, we also find tissue-specific patterns for sites under neutral evolution and for positive selection. We observe fast evolution of genes expressed in testis, but also in other tissues, notably liver, which are explained by weak purifying selection rather than by positive selection. PMID:26121354
Complexity and multifractality of neuronal noise in mouse and human hippocampal epileptiform dynamics
NASA Astrophysics Data System (ADS)
Serletis, Demitre; Bardakjian, Berj L.; Valiante, Taufik A.; Carlen, Peter L.
Fractal methods offer an invaluable means of investigating turbulent nonlinearity in non-stationary biomedical recordings from the brain. Here, we investigate properties of complexity (i.e. the correlation dimension, maximum Lyapunov exponent, 1/f? noise and approximate entropy) and multifractality in background neuronal noise-like activity underlying epileptiform transitions recorded at the intracellular and local network scales from two in vitro models: the whole-intact mouse hippocampus and lesional human hippocampal slices. Our results show evidence for reduced dynamical complexity and multifractal signal features following transition to the ictal epileptiform state. These findings suggest that pathological breakdown in multifractal complexity coincides with loss of signal variability or heterogeneity, consistent with an unhealthy ictal state that is far from the equilibrium of turbulent yet healthy fractal dynamics in the brain. Thus, it appears that background noise-like activity successfully captures complex and multifractal signal features that may, at least in part, be used to classify and identify brain state transitions in the healthy and epileptic brain, offering potential promise for therapeutic neuromodulatory strategies for afflicted patients suffering from epilepsy and other related neurological disorders. This paper is based on chapter 5 of Serletis (2010 PhD Dissertation Department of Physiology, Institute of Biomaterials and Biomedical Engineering, University of Toronto).
Derivation and Expansion of PAX7-Positive Muscle Progenitors from Human and Mouse Embryonic Stem Cells
Shelton, Michael; Metz, Jeff; Liu, Jun; Carpenedo, Richard�L.; Demers, Simon-Pierre; Stanford, William�L.; Skerjanc, Ilona�S.
Summary Cell therapies treating pathological muscle atrophy or damage requires an adequate quantity of muscle progenitor cells (MPCs) not currently attainable from adult donors. Here, we generate cultures of approximately 90% skeletal myogenic cells by treating human embryonic stem cells (ESCs) with the GSK3 inhibitor CHIR99021 followed by FGF2 and N2 supplements. Gene expression analysis identified progressive expression of mesoderm, somite, dermomyotome, and myotome markers, following patterns of embryonic myogenesis. CHIR99021 enhanced transcript levels of the pan-mesoderm gene T and paraxial-mesoderm genes MSGN1 and TBX6; immunofluorescence confirmed that 91% � 6% of cells expressed T immediately following treatment. By 7�weeks, 47% � 3% of cells were MYH+ve myocytes/myotubes surrounded by a 43% � 4% population of PAX7+ve MPCs, indicating 90% of cells had achieved myogenic identity without any cell sorting. Treatment of mouse ESCs with these factors resulted in similar enhancements of myogenesis. These studies establish a foundation for serum-free and chemically defined monolayer skeletal myogenesis of ESCs. PMID:25241748
Elimination of human leukemia by monoclonal antibodies in an athymic nude mouse leukemia model.
Xu, Y; Scheinberg, D A
A human acute myeloid leukemia model has been developed by i.v. transplantation of HL-60 myeloid leukemia cells into Swiss nude mice pretreated with cyclophosphamide. HL-60 cells disseminated into hematopoietic tissues as determined by flow cytometric analysis, fluorescence microscopy, fluorescence in situ hybridization analysis, and colony formation assay. Passive immunotherapy using murine anti-CD13 (F23) or anti-CD33 (M195) mAbs was able to eliminate completely the HL-60 cells in the mice, as determined by fluorescence in situ hybridization analysis, colony formation assay, and culture of mouse blood and tissue cells in vitro. Although F23 is able to inhibit completely CD13/aminopeptidase N enzymatic activity, actinonin, another potent inhibitor of CD13/aminopeptidase N, was not active as an antileukemic agent. HL-60 cell surface antigens, including CD13 (aminopeptidase N) and CD33 (p67), down-regulated over time, and murine anti-HL-60 antibody was generated while the cells grew in the mice. This response was suppressed by cyclophosphamide. These data suggest that leukemia cell elimination was antibody mediated. PMID:9815910
Intracellular distribution of Fe3O4 nanoparticles in both human and mouse cells
NASA Astrophysics Data System (ADS)
Palihawadana Arachchige, Maheshika; Laha, Suvra; Rajagopal, Amulya; Kulkarni, Sanjana; Wang, Shuo; Flack, Amanda; Li, Chunying; Jena, Bhanu; Lawes, Gavin
In recent years there has been an increasing interest in developing Fe3O4 nanoparticles for biomedical applications including targeted drug delivery and magnetic resonance imaging. Understanding of the intracellular distribution of these nanoparticles is crucial when considering these nanoparticles for specific applications. We have synthesized Fe3O4 nanoparticles having average size of 14 nm using a co-precipitation technique, which were coated with dextran. We studied the structural and morphological characteristics of the nanoparticles using x-ray diffraction, electron microscopy, dynamic light scattering, and zeta potential measurements. We also characterized the magnetic properties of the nanoparticles. In order to investigate the intracellular distribution of these Fe3O4 nanoparticles, we functionalized the dextran coated Fe3O4 nanoparticles with a fluorescent dye, Fluorescein isothiocyanate (FITC), and cultured them with both mouse insulinoma MIN 6 cells and human pancreatic MIA PaCa 2 cells. Using optical microscope we investigated the intracellular distribution of the nanoparticles and the effects on cell growth.
Guiding the osteogenic fate of mouse and human mesenchymal stem cells through feedback system control
Honda, Yoshitomo; Ding, Xianting; Mussano, Federico; Wiberg, Akira; Ho, Chih-ming; Nishimura, Ichiro
Stem cell-based disease modeling presents unique opportunities for mechanistic elucidation and therapeutic targeting. The stable induction of fate-specific differentiation is an essential prerequisite for stem cell-based strategy. Bone morphogenetic protein 2 (BMP-2) initiates receptor-regulated Smad phosphorylation, leading to the osteogenic differentiation of mesenchymal stromal/stem cells (MSC) in vitro; however, it requires supra-physiological concentrations, presenting a bottleneck problem for large-scale drug screening. Here, we report the use of a double-objective feedback system control (FSC) with a differential evolution (DE) algorithm to identify osteogenic cocktails of extrinsic factors. Cocktails containing significantly reduced doses of BMP-2 in combination with physiologically relevant doses of dexamethasone, ascorbic acid, beta-glycerophosphate, heparin, retinoic acid and vitamin D achieved accelerated in vitro mineralization of mouse and human MSC. These results provide insight into constructive approaches of FSC to determine the applicable functional and physiological environment for MSC in disease modeling, drug screening and tissue engineering. PMID:24305548
Expression of the human apolipoprotein E gene suppresses steroidogenesis in mouse Y1 adrenal cells
Reyland, M.E.; Forgez, P.; Prack, M.M.; Williams, D.L. (State Univ. of New York, Stony Brook (United States)); Gwynne, J.T. (Univ. of North Carolina, Chapel Hill (United States))
The lipid transport protein, apolipoprotein E (apoE), is expressed in many peripheral tissues in vivo including the adrenal gland and testes. To investigate the role of apoE in adrenal cholesterol homeostasis, the authors have expressed a human apoE genomic clone in the Y1 mouse adrenocortical cell line. Y1 cells do not express endogenous apoE mRNA or protein. Expression of apoE in Y1 cells resulted in a dramatic decrease in basal steroidogenesis; secretion of fluorogenic steroid was reduced 7- to {gt}100-fold relative to Y1 parent cells. Addition of 5-cholesten-3{beta},25-idol failed to overcome the suppression of steroidogenesis in these cells. Cholesterol esterification under basal conditions, as measured by the production of cholesteryl ({sup 14}C)oleate, was similar in the Y1 parent and the apoE-transfected cell lines. Upon incubation with adrenocorticotropin or dibutyryl cAMP, production of cholesteryl ({sup 14}C)oleate decreased 5-fold in the Y1 parent cells but was unchanged in the apoE-transfected cell lines. These results suggest that apoE may be an important modulator of cholesterol utilization and steroidogenesis in adrenal cells.
MUC1 Selectively Targets Human Pancreatic Cancer in Orthotopic Nude Mouse Models
Park, Jeong Youp; Hiroshima, Yukihiko; Lee, Jin Young; Maawy, Ali A.; Hoffman, Robert M.; Bouvet, Michael
The goal of this study was to determine whether MUC1 antibody conjugated with a fluorophore could be used to visualize pancreatic cancer. Anti-MUC1 (CT2) antibody was conjugated with 550 nm or 650 nm fluorophores. Nude mouse were used to make subcutaneous and orthotopic models of pancreatic cancer. Western blot and flow cytometric analysis confirmed the expression of MUC1 in human pancreatic cancer cell lines including BxPC-3 and Panc-1. Immunocytochemistry with fluorophore conjugated anti-MUC1 antibody demonstrated fluorescent areas on the membrane of Panc-1 cancer cells. After injecting the conjugated anti-MUC1 antibodies via the tail vein, subcutaneously transplanted Panc-1 and BxPC-3 tumors emitted strong fluorescent signals. In the subcutaneous tumor models, the fluorescent signal from the conjugated anti-MUC1 antibody was noted around the margin of the tumor and space between the cells. The conjugated anti-MUC1 antibody bound the tumor in orthotopically-transplanted Panc-1 and BxPC-3 models enabling the tumors to be imaged. This study showed that fluorophore conjugated anti-MUC1 antibodies could visualize pancreatic tumors in vitro and in vivo and may help to improve the diagnosis and treatment of pancreatic cancer. PMID:25815753
Mouse and human islets survive and function after coating by biosilicification
Jaroch, David B.; Lu, Jing; Madangopal, Rajtarun; Stull, Natalie D.; Stensberg, Matthew; Shi, Jin; Kahn, Jennifer L.; Herrera-Perez, Ruth; Zeitchek, Michael; Sturgis, Jennifer; Robinson, J. Paul; Yoder, Mervin C.; Porterfield, D. Marshall; Mirmira, Raghavendra G.
Inorganic materials have properties that can be advantageous in bioencapsulation for cell transplantation. Our aim was to engineer a hybrid inorganic/soft tissue construct by inducing pancreatic islets to grow an inorganic shell. We created pancreatic islets surrounded by porous silica, which has potential application in the immunoprotection of islets in transplantation therapies for type 1 diabetes. The new method takes advantage of the islet capsule surface as a template for silica formation. Mouse and human islets were exposed to medium containing saturating silicic acid levels for 9�15 min. The resulting tissue constructs were then cultured for up to 4 wk under normal conditions. Scanning electron microscopy and energy dispersive X-ray spectroscopy was used to monitor the morphology and elemental composition of the material at the islet surface. A cytokine assay was used to assess biocompatibility with macrophages. Islet survival and function were assessed by confocal microscopy, glucose-stimulated insulin release assays, oxygen flux at the islet surface, expression of key genes by RT-PCR, and syngeneic transplant into diabetic mice. PMID:24002572
Functional recovery with recombinant human IGF1 treatment in a mouse model of Rett Syndrome.
Castro, Jorge; Garcia, Rodrigo I; Kwok, Showming; Banerjee, Abhishek; Petravicz, Jeremy; Woodson, Jonathan; Mellios, Nikolaos; Tropea, Daniela; Sur, Mriganka
Rett Syndrome is a neurodevelopmental disorder that arises from mutations in the X-linked gene methyl-CpG binding protein 2 (MeCP2). MeCP2 has a large number of targets and a wide range of functions, suggesting the hypothesis that functional signaling mechanisms upstream of synaptic and circuit maturation may contribute to our understanding of the disorder and provide insight into potential treatment. Here, we show that insulin-like growth factor-1 (IGF1) levels are reduced in young male Mecp2-null (Mecp2(-/y)) mice, and systemic treatment with recombinant human IGF1 (rhIGF1) improves lifespan, locomotor activity, heart rate, respiration patterns, and social and anxiety behavior. Furthermore, Mecp2-null mice treated with rhIGF1 show increased synaptic and activated signaling pathway proteins, enhanced cortical excitatory synaptic transmission, and restored dendritic spine densities. IGF1 levels are also reduced in older, fully symptomatic heterozygous (Mecp2(-/+)) female mice, and short-term treatment with rhIGF1 in these animals improves respiratory patterns, reduces anxiety levels, and increases exploratory behavior. In addition, rhIGF1 treatment normalizes abnormally prolonged plasticity in visual cortex circuits of adult Mecp2(-/+) female mice. Our results provide characterization of the phenotypic development of Rett Syndrome in a mouse model at the molecular, circuit, and organismal levels and demonstrate a mechanism-based therapeutic role for rhIGF1 in treating Rett Syndrome. PMID:24958891
Induction of apoptotic cell death in mouse lymphoma and human leukemia cell lines by a calcium-binding protein complex, calprotectin, derived from inflammatory peritoneal exudate cells
Satoru Yui; Masaaki Mikami; Masatoshi Yamazaki
We have previously shown that the cal- cium-binding protein complex, caiprotectin, purified from rat inflammatory peritoneal cells exerts marked cytotoxic activity against rat, mouse, and human tumor cells. We studied here whether the cytotoxicity is caused by induction of apoptosis, using mouse EL-4 lymphoma and human MOLT-4 leukemia lines as targets. The rat caiprotectin sample inhibited (3H)thymidine incorporation into these
A Non-Leaky Artemis-Deficient Mouse That Accurately Models the Human Severe Combined Immune Deficiency Phenotype, Including Resistance to Hematopoietic Stem Cell Transplantation
Zheng Xiao; Elizabeth Dunn; Kanal Singh; Imran S. Khan; Steven M. Yannone; Morton J. Cowan
Two Artemis-deficient (mArt-\\/-) mouse models, generated independently on 129\\/SvJ backgrounds, have the expected T-B-NK+ severe combined immune deficiency (SCID) phenotype but fail to mimic the human disease because of CD4+ T cell leakiness. Moreover, immune reconstitution after hematopoietic stem cell transplantation is achieved more readily in these leaky mouse models than in Artemis-deficient humans. To develop a more clinically relevant
Lipid characterization of mouse and human fibroblasts and their SV 40-transformed analogues: effects of supplementation with 20alpha hydrocholesterol, linoleic acid, and Sterculia foetida seed oil�
Pierce-Ruhland, Richard Arthur
-RUHLAND Submitted to the Graduate Co11ege of Texas ARM University i n parti a1 fuifi 1 lment of the requirement for the deoree of MASTER OF SCIENCE December 198', Major Subject: Biochemistry LIPID CHARACTERIZATION OF MOUSE AND HUMAN FIBROBLASTS AND THEIR SV40... of Department) I December 1981 ABSTRACT Lipid Characterization of Mouse and Human Fibroblasts and Their SV40-Transformed Analogues: Effects of Supplementation with 20m Hydroxycholesterol, Linoleic Acid, and Sterculia foetida Seed Oil. (December 1981...
Mouse Autosomal Homolog of DAZ,a Candidate Male Sterility Gene in Humans, Is Expressed in Male Germ Cells before and after Puberty
Renee Reijo; Judith Seligman; Mary Beth Dinulos; Tom Jaffe; Laura G. Brown; Christine M. Disteche; David C. Page
Deletion of theAzoospermia Factor(AZF) region of the human Y chromosome results in spermatogenic failure. While the identity of the critical missing gene has yet to be established, a strong candidate is the putative RNA-binding protein DAZ (Deleted in Azoospermia). Here we describe the mouse homolog of DAZ. Unlike humanDAZ,which is Y-linked, in mouse theDazh(DAZ homolog) gene maps to chromosome 17.
Headbobber: A Combined Morphogenetic and Cochleosaccular Mouse Model to Study 10qter Deletions in Human Deafness
Buniello, Annalisa; Hardisty-Hughes, Rachel E.; Pass, Johanna C.; Bober, Eva; Smith, Richard J.; Steel, Karen P.
The recessive mouse mutant headbobber (hb) displays the characteristic behavioural traits associated with vestibular defects including headbobbing, circling and deafness. This mutation was caused by the insertion of a transgene into distal chromosome 7 affecting expression of native genes. We show that the inner ear of hb/hb mutants lacks semicircular canals and cristae, and the saccule and utricle are fused together in a single utriculosaccular sac. Moreover, we detect severe abnormalities of the cochlear sensory hair cells, the stria vascularis looks severely disorganised, Reissner's membrane is collapsed and no endocochlear potential is detected. Myo7a and Kcnj10 expression analysis show a lack of the melanocyte-like intermediate cells in hb/hb stria vascularis, which can explain the absence of endocochlear potential. We use Trp2 as a marker of melanoblasts migrating from the neural crest at E12.5 and show that they do not interdigitate into the developing strial epithelium, associated with abnormal persistence of the basal lamina in the hb/hb cochlea. We perform array CGH, deep sequencing as well as an extensive expression analysis of candidate genes in the headbobber region of hb/hb and littermate controls, and conclude that the headbobber phenotype is caused by: 1) effect of a 648 kb deletion on distal Chr7, resulting in the loss of three protein coding genes (Gpr26, Cpmx2 and Chst15) with expression in the inner ear but unknown function; and 2) indirect, long range effect of the deletion on the expression of neighboring genes on Chr7, associated with downregulation of Hmx3, Hmx2 and Nkx1.2 homeobox transcription factors. Interestingly, deletions of the orthologous region in humans, affecting the same genes, have been reported in nineteen patients with common features including sensorineural hearing loss and vestibular problems. Therefore, we propose that headbobber is a useful model to gain insight into the mechanisms underlying deafness in human 10qter deletion syndrome. PMID:23457544
Xenotransplantation of human cultured parathyroid progenitor cells into mouse peritoneum does not induce rejection reaction
Wo?niewicz, Bogdan; Szmidt, Jacek; ?ladowski, Dariusz; Zaj?c, Krzysztof; Chudzi?ski, Witold
Introduction Parathyroid progenitor cells devoid of immunogenic antigens were used for human allotransplantation. Although there were many potential reasons for the expiry of transplant activity in humans, we decided to exclude a subclinical form of rejection reaction, and test the rejection reaction in an animal model. Material and methods Experiments were carried out on 40 conventional male mice in their third month of life. The animals were housed in groups of 10 per cage in 4 cages with fitted water dispensers and fed a conventional diet based on standard pellet food. They were divided into four groups of 10 animals each, three experimental groups and one control group. Identified progenitor cells were stored in a cell bank. After testing the phenotype, viability, and absence of immunogenic properties, the cells were transplanted into mouse peritoneum cavity. Results Animals were observed for 9 weeks. At 9 weeks of observation, the mean serum PTH concentration in the experimental groups was 2.0-2.5 pg/ml, while in the control group it did not exceed 1.5 pg/ml. The immunohistochemical assays demonstrated that millions of viable cells with a phenotype identical to the endocrine cells had survived in the peritoneum. Histologic specimens from different internal organs stained for PTH revealed positive cells labelled with anti-PTH Ab in the intestinal lamina, brain, liver, and spleen. Conclusions In the present paper we have demonstrated that xenotransplantation may be used as a model for an explanation of the immunogenic properties of cells generated from postnatal organs for regenerative therapy.
Neuron-Enriched Gene Expression Patterns are Regionally Anti-Correlated with Oligodendrocyte-Enriched Patterns in the Adult Mouse and Human Brain
Tan, Powell Patrick Cheng; French, Leon; Pavlidis, Paul
An important goal in neuroscience is to understand gene expression patterns in the brain. The recent availability of comprehensive and detailed expression atlases for mouse and human creates opportunities to discover global patterns and perform cross-species comparisons. Recently we reported that the major source of variation in gene transcript expression in the adult normal mouse brain can be parsimoniously explained as reflecting regional variation in glia to neuron ratios, and is correlated with degree of connectivity and location in the brain along the anterior-posterior axis. Here we extend this investigation to two gene expression assays of adult normal human brains that consisted of over 300 brain region samples, and perform comparative analyses of brain-wide expression patterns to the mouse. We performed principal components analysis (PCA) on the regional gene expression of the adult human brain to identify the expression pattern that has the largest variance. As in the mouse, we observed that the first principal component is composed of two anti-correlated patterns enriched in oligodendrocyte and neuron markers respectively. However, we also observed interesting discordant patterns between the two species. For example, a few mouse neuron markers show expression patterns that are more correlated with the human oligodendrocyte-enriched pattern and vice-versa. In conclusion, our work provides insights into human brain function and evolution by probing global relationships between regional cell type marker expression patterns in the human and mouse brain. PMID:23440889
Cloning and sequence comparison of the mouse, human, and chicken engrailed genes reveal potential functional domains and regulatory regions.
Logan, C; Hanks, M C; Noble-Topham, S; Nallainathan, D; Provart, N J; Joyner, A L
We have isolated and characterized genomic DNA clones for the human and chicken homologues of the mouse En-1 and En-2 genes and determined the genomic structure and predicted protein sequences of both En genes in all three species. Comparison of these vertebrate En sequences with the Xenopus En-2 [Hemmati-Brivanlou et al., 1991) and invertebrate engrailed-like genes showed that the two previously identified highly conserved regions within the En protein ]reviewed in Joyner and Hanks, 1991] can be divided into five distinct subregions, designated EH1 to EH5. Sequences 5' and 3' to the predicted coding regions of the vertebrate En genes were also analyzed in an attempt to identify cis-acting DNA sequences important for the regulation of En gene expression. Considerable sequence similarity was found between the mouse and human homologues both within the putative 5' and 3' untranslated as well as 5' flanking regions. Between the mouse and Xenopus En-2 genes, shorter stretches of sequence similarity were found within the 3' untranslated region. The 5' untranslated regions of the mouse, chicken and Xenopus En-2 genes, however, showed no similarly conserved stretches. In a preliminary analysis of the expression pattern of the human En genes, En-2 protein and RNA were detected in the embryonic and adult cerebellum respectively and not in other tissues tested. These patterns are analogous to those seen in other vertebrates. Taken together these results further strengthen the suggestion that En gene function and regulation has been conserved throughout vertebrate evolution and, along with the five highly conserved regions within the En protein, raise an interesting question about the presence of conserved genetic pathways. PMID:1363401
Differences in amyloid-? clearance across mouse and human blood-brain barrier models: Kinetic analysis and mechanistic modeling
Qosa, Hisham; Abuasal, Bilal S.; Romero, Ignacio A.; Weksler, Babette; Couraud, Pierre-Oliver; Keller, Jeffrey N.; Kaddoumi, Amal
Alzheimer�s disease (AD) has a characteristic hallmark of amyloid-? (A?) accumulation in the brain. This accumulation of A? has been related to its faulty cerebral clearance. Indeed, preclinical studies that used mice to investigate A? clearance showed that efflux across blood-brain barrier (BBB) and brain degradation mediate efficient A? clearance. However, the contribution of each process to A? clearance remains unclear. Moreover, it is still uncertain how species differences between mouse and human could affect A? clearance. Here, a modified form of the brain efflux index method was used to estimate the contribution of BBB and brain degradation to A? clearance from the brain of wild type mice. We estimated that 62% of intracerebrally injected 125I-A?40 is cleared across BBB while 38% is cleared by brain degradation. Furthermore, in vitro and in silico studies were performed to compare A? clearance between mouse and human BBB models. Kinetic studies for A?40 disposition in bEnd3 and hCMEC/D3 cells, representative in vitro mouse and human BBB models, respectively, demonstrated 30-fold higher rate of 125I-A?40 uptake and 15-fold higher rate of degradation by bEnd3 compared to hCMEC/D3 cells. Expression studies showed both cells to express different levels of P-glycoprotein and RAGE, while LRP1 levels were comparable. Finally, we established a mechanistic model, which could successfully predict cellular levels of 125I-A?40 and the rate of each process. Established mechanistic model suggested significantly higher rates of A? uptake and degradation in bEnd3 cells as rationale for the observed differences in 125I-A?40 disposition between mouse and human BBB models. In conclusion, current study demonstrates the important role of BBB in the clearance of A? from the brain. Moreover, it provides insight into the differences between mouse and human BBB with regards to A? clearance and offer, for the first time, a mathematical model that describes A? clearance across BBB. PMID:24467845
Ventriculomegaly associated with ependymal gliosis and declines in barrier integrity in the aging human and mouse brain.
Shook, Brett A; Lennington, Jessica B; Acabchuk, Rebecca L; Halling, Meredith; Sun, Ye; Peters, John; Wu, Qian; Mahajan, Amit; Fellows, Douglas W; Conover, Joanne C
Age-associated ventriculomegaly is typically attributed to neurodegeneration; however, additional factors might initiate or contribute to progressive ventricular expansion. By directly linking postmortem human MRI sequences with histological features of periventricular tissue, we show that substantial lateral ventricle surface gliosis is associated with ventriculomegaly. To examine whether loss of ependymal cell coverage resulting in ventricle surface glial scarring can lead directly to ventricle enlargement independent of any other injury or degenerative loss, we modeled in mice the glial scarring found along the lateral ventricle surface in aged humans. Neuraminidase, which cleaves glycosidic linkages of apical adherens junction proteins, was administered intracerebroventricularly to denude areas of ependymal cells. Substantial ependymal cell loss resulted in reactive gliosis rather than stem cell-mediated regenerative repair of the ventricle lining, and the gliotic regions showed morphologic and phenotypic characteristics similar to those found in aged humans. Increased levels of aquaporin-4, indicative of edema, observed in regions of periventricular gliosis in human tissue were also replicated in our mouse model. 3D modeling together with volume measurements revealed that mice with ventricle surface scarring developed expanded ventricles, independent of neurodegeneration. Through a comprehensive, comparative analysis of the lateral ventricles and associated periventricular tissue in aged humans and mouse, followed by modeling of surface gliosis in mice, we have demonstrated a direct link between lateral ventricle surface gliosis and ventricle enlargement. These studies highlight the importance of maintaining an intact ependymal cell lining throughout aging. PMID:24341850
Effect of mono-(2-ethylhexyl) phthalate on human and mouse fetal testis: In vitro and in vivo approaches
Muczynski, V. [Univ. Paris Diderot, Sorbonne Paris Cit�, Laboratory of Development of the Gonads, Unit of Stem Cells and Radiation, BP 6, 92265 Fontenay-aux-Roses (France) [Univ. Paris Diderot, Sorbonne Paris Cit�, Laboratory of Development of the Gonads, Unit of Stem Cells and Radiation, BP 6, 92265 Fontenay-aux-Roses (France); CEA, DSV, iRCM, SCSR, LDRG, 92265 Fontenay-aux-Roses (France); INSERM, Unit� 967, F-92265, Fontenay aux Roses (France); Cravedi, J.P. [INRA, INP, Universit� de Toulouse, UMR1331 TOXALIM, F-31027, Toulouse (France)] [INRA, INP, Universit� de Toulouse, UMR1331 TOXALIM, F-31027, Toulouse (France); Lehraiki, A.; Levacher, C.; Moison, D.; Lecureuil, C.; Messiaen, S. [Univ. Paris Diderot, Sorbonne Paris Cit�, Laboratory of Development of the Gonads, Unit of Stem Cells and Radiation, BP 6, 92265 Fontenay-aux-Roses (France) [Univ. Paris Diderot, Sorbonne Paris Cit�, Laboratory of Development of the Gonads, Unit of Stem Cells and Radiation, BP 6, 92265 Fontenay-aux-Roses (France); CEA, DSV, iRCM, SCSR, LDRG, 92265 Fontenay-aux-Roses (France); INSERM, Unit� 967, F-92265, Fontenay aux Roses (France); Perdu, E. [INRA, INP, Universit� de Toulouse, UMR1331 TOXALIM, F-31027, Toulouse (France)] [INRA, INP, Universit� de Toulouse, UMR1331 TOXALIM, F-31027, Toulouse (France); Frydman, R. [Service de Gyn�cologie-Obst�trique, H�pital A. B�cl�re, Universit� Paris Sud F-92141 Clamart (France)] [Service de Gyn�cologie-Obst�trique, H�pital A. B�cl�re, Universit� Paris Sud F-92141 Clamart (France); Habert, R. [Univ. Paris Diderot, Sorbonne Paris Cit�, Laboratory of Development of the Gonads, Unit of Stem Cells and Radiation, BP 6, 92265 Fontenay-aux-Roses (France) [Univ. Paris Diderot, Sorbonne Paris Cit�, Laboratory of Development of the Gonads, Unit of Stem Cells and Radiation, BP 6, 92265 Fontenay-aux-Roses (France); CEA, DSV, iRCM, SCSR, LDRG, 92265 Fontenay-aux-Roses (France); INSERM, Unit� 967, F-92265, Fontenay aux Roses (France); and others
The present study was conducted to determine whether exposure to the mono-(2-ethylhexyl) phthalate (MEHP) represents a genuine threat to male human reproductive function. To this aim, we investigated the effects on human male fetal germ cells of a 10{sup ?5} M exposure. This dose is slightly above the mean concentrations found in human fetal cord blood samples by biomonitoring studies. The in vitro experimental approach was further validated for phthalate toxicity assessment by comparing the effects of in vitro and in vivo exposure in mouse testes. Human fetal testes were recovered during the first trimester (7�12 weeks) of gestation and cultured in the presence or not of 10{sup ?5} M MEHP for three days. Apoptosis was quantified by measuring the percentage of Caspase-3 positive germ cells. The concentration of phthalate reaching the fetal gonads was determined by radioactivity measurements, after incubations with {sup 14}C-MEHP. A 10{sup ?5} M exposure significantly increased the rate of apoptosis in human male fetal germ cells. The intratesticular MEHP concentration measured corresponded to the concentration added in vitro to the culture medium. Furthermore, a comparable effect on germ cell apoptosis in mouse fetal testes was induced both in vitro and in vivo. This study suggests that this 10{sup ?5} M exposure is sufficient to induce changes to the in vivo development of the human fetal male germ cells. -- Highlights: ? 10{sup ?5} M of MEHP impairs germ cell development in the human fetal testis. ? Organotypic culture is a suitable approach to investigate phthalate effects in human. ? MEHP is not metabolized in the human fetal testis. ? In mice, MEHP triggers similar effects both in vivo and in vitro.
Ano1 is a selective marker of interstitial cells of Cajal in the human and mouse gastrointestinal tract.
Gomez-Pinilla, Pedro J; Gibbons, Simon J; Bardsley, Michael R; Lorincz, Andrea; Pozo, Maria J; Pasricha, Pankaj J; Van de Rijn, Matt; West, Robert B; Sarr, Michael G; Kendrick, Michael L; Cima, Robert R; Dozois, Eric J; Larson, David W; Ordog, Tamas; Farrugia, Gianrico
Populations of interstitial cells of Cajal (ICC) are altered in several gastrointestinal neuromuscular disorders. ICC are identified typically by ultrastructure and expression of Kit (CD117), a protein that is also expressed on mast cells. No other molecular marker currently exists to independently identify ICC. The expression of ANO1 (DOG1, TMEM16A), a Ca(2+)-activated Cl(-) channel, in gastrointestinal stromal tumors suggests it may be useful as an ICC marker. The aims of this study were therefore to determine the distribution of Ano1 immunoreactivity compared with Kit and to establish whether Ano1 is a reliable marker for human and mouse ICC. Expression of Ano1 in human and mouse stomach, small intestine, and colon was investigated by immunofluorescence labeling using antibodies to Ano1 alone and in combination with antibodies to Kit. Colocalization of immunoreactivity was demonstrated by epifluorescence and confocal microscopy. In the muscularis propria, Ano1 immunoreactivity was restricted to cells with the morphology and distribution of ICC. All Ano1-positive cells in the muscularis propria were also Kit positive. Kit-expressing mast cells were not Ano1 positive. Some non-ICC in the mucosa and submucosa of human tissues were Ano1 positive but Kit negative. A few (3.2%) Ano1-positive cells in the human gastric muscularis propria were labeled weakly for Kit. Ano1 labels all classes of ICC and represents a highly specific marker for studying the distribution of ICC in mouse and human tissues with an advantage over Kit since it does not label mast cells. PMID:19372102
Human Hair Follicle Cells with the Cell Surface Marker CD34 Can Regenerate New Mouse Hair Follicles and Located in the Outer Root Sheath of Immunodeficient Nude Mice
Lee, Sung-Hoon; Chung, Mi-Kyung; Sohn, Yong-Jin; Lee, Yong-Soon; Kang, Kyung-Sun
Background and Objectives: The bulge region of hair follicle has been reported as a putative reservoir of hair follicle stem cells. The purpose of this study was to compare hair follice CD34 negative (CD34?) cell with CD34 positive (CD34+) cell and to evaluate the ability to regenerate new hair of immunodeficient nude mouse. Methods and Results: In this report, we isolated the cells with CD34, known as bulge-negative cell surface marker from cultured human hair follicle cells using by magnetic cell sorting (MACS), injected the cells to immunodeficient nude mouse. To determine immunological characterization, human hair follicle CD34+ cells and CD34? cells were assessed by flow cytometry. The localization of injected-CD34+ cells was assessed on formalin-fixed, paraffin-embedded mouse skin samples by in situ hybridization technique. Our findings show that the human hair follicle cells with cell surface marker CD34 were located in the outer root sheath of nude mouse after transplantation and the cells were able to regenerate new hair follicle in immunodeficient nude mouse. CD34? cells also were able to regenerate follicles in the mouse, however, CD34+ cells were able to regenerate much more hair follicle than CD34? cells. Conclusions: Therefore, the results of this study add new insight into the investigation of CD34 stem cell-related molecule in human hair follicles and suggest that not all human hair follicle stem cells reside in bulge region, but in a lager niche. PMID:24855511
Molecular cloning and chromosomal assignment of the mouse C-type natriuretic peptide (CNP) gene (Nppc): Comparison with the human CNP gene (NPPC)
Yoshihiro, Ogawa; Hiroshi, Itoh; Yuka, Yoshitake [Kyoto Univ. (Japan)] [and others
The mouse C-type natriuretic peptide (CNP) genomic fragment was isolated from a mouse genomic DNA library. The mouse CNP gene is composed of at least two exons and one intron. The 5{prime}-flanking region contains an array of cis-acting regulatory elements and a dinucleotide CA repeat (microsatellite). Analysis of the deduced amino acid sequences revealed that mouse preproCNP is a 126-amino-acid peptide and that its C-terminal 22-residue peptide preceded by Lys-Lys is identical to porcine, rat, and human CNPs. On the basis of the polymerase chain reaction-analyzed microsatellite length polymorphisms among recombinant inbred strains of mice, the CNP gene (Nppc) was assigned to mouse chromosome 1. Furthermore, the human CNP 5{prime}-flanking region was extended for sequencing, and comparison of the mouse and human CNP genomic sequences revealed regions of conservation and diversity. Using somatic hybrid cell methodology, the CNP gene (NPPC) was assigned to human chromosome 2. The present study has added another locus to the conserved syntenic group in mice and humans. 1 fig., 1 tab.
Cloning of the mouse hepatitis virus (MHV) receptor: expression in human and hamster cell lines confers susceptibility to MHV.
Dveksler, G S; Pensiero, M N; Cardellichio, C B; Williams, R K; Jiang, G S; Holmes, K V; Dieffenbach, C W
The cellular receptor for murine coronavirus mouse hepatitis virus (MHV)-A59 is a member of the carcinoembryonic antigen (CEA) family of glycoproteins in the immunoglobulin superfamily. We isolated a cDNA clone (MHVR1) encoding the MHV receptor. The sequence of this clone predicts a 424-amino-acid glycoprotein with four immunoglobulinlike domains, a transmembrane domain, and a short intracytoplasmic tail, MHVR1 is closely related to the murine CEA-related clone mmCGM1 (Mus musculus carcinoembryonic antigen gene family member). Western blot (immunoblot) analysis performed with antireceptor antibodies detected a glycoprotein of 120 kDa in BHK cells stably transfected with MHVR1. This corresponds to the size of the MHV receptor expressed in mouse intestine and liver. Human and hamster fibroblasts transfected with MHVR1 became susceptible to infection with MHV-A59. Like MHV-susceptible mouse fibroblasts, the MHVR1-transfected human and hamster cells were protected from MHV infection by pretreatment with monoclonal antireceptor antibody CC1. Thus, the 110- to 120-kDa CEA-related glycoprotein encoded by MHVR1 is a functional receptor for murine coronavirus MHV-A59. PMID:1719235
Identification of Novel Alternative Splice Isoforms of Circulating Proteins in a Mouse Model of Human Pancreatic Cancer
Menon, Rajasree; Zhang, Qing; Zhang, Yan; Fermin, Damian; Bardeesy, Nabeel; DePinho, Ronald A.; Lu, Chunxia; Hanash, Samir M.; Omenn, Gilbert S.; States, David J.
To assess the potential of tumor-associated alternatively spliced gene products as a source of biomarkers in biological fluids, we have analyzed a large dataset of mass spectra derived from the plasma proteome of a mouse model of human pancreatic ductal adenocarcinoma. MS/MS spectra were interrogated for novel splice isoforms using a non-redundant database containing an exhaustive 3-frame translation of Ensembl transcripts and gene models from ECgene. This integrated analysis identified 420 distinct splice isoforms, of which 92 did not match any previously annotated mouse protein sequence. We chose seven of those novel variants for validation by reverse transcription polymerase chain reaction (RT-PCR). The results were concordant with the proteomic analysis. All seven novel peptides were successfully amplified in pancreas specimens from both wild-type and mutant mice. Isotopic labeling of cysteine-containing peptides from tumor-bearing mice and wild-type controls enabled relative quantification of the proteins. Differential expression between tumor-bearing and control mice was notable for peptides from novel variants of muscle pyruvate kinase, malate dehydrogenase 1, glyceraldehyde-3-phosphate dehydrogenase, proteoglycan 4, minichromosome maintenance, complex component 9, high mobility group box 2 and hepatocyte growth factor activator. Our results show that, in a mouse model for human pancreatic cancer, novel and differentially expressed alternative splice isoforms are detectable in plasma and may be a source of candidate biomarkers. PMID:19118015
Animal models of human prostate cancer: The Consensus Report of the New York Meeting of the Mouse Models of Human Cancers Consortium Prostate Pathology Committee
Ittmann, Michael; Huang, Jiaoti; Radaelli, Enrico; Martin, Philip; Signoretti, Sabina; Sullivan, Ruth; Simons, Brian W.; Ward, Jerrold M.; Robinson, Brian D.; Chu, Gerald C.; Loda, Massimo; Thomas, George; Borowsky, Alexander; Cardiff, Robert D.
Animal models, particularly mouse models, play a central role in the study of the etiology, prevention and treatment of human prostate cancer (PCa). While tissue culture models are extremely useful in understanding the biology of PCa, they cannot recapitulate the complex cellular interactions within the tumor microenvironment that play a key role in cancer initiation and progression. The NCI Mouse Models of Human Cancers Consortium convened a group of human and veterinary pathologists to review the current animal models of PCa and make recommendations regarding the pathological analysis of these models. Over 40 different models with 439 samples were reviewed including genetically engineered mouse models, xenograft, rat and canine models. Numerous relevant models have been developed over the last 15 years and each approach has strengths and weaknesses. Analysis of multiple genetically engineered models has shown that reactive stroma formation is present in all the models developing invasive carcinomas. In addition, numerous models with multiple genetic alterations display aggressive phenotypes characterized by sarcomatoid carcinomas and metastases, which is presumably a histological manifestation of epithelial-mesenchymal transition. The significant progress in development of improved models of PCa has already accelerated our understanding the complex biology of PCa and promises to enhance development of new approaches to prevention, detection and treatment of this common malignancy. PMID:23610450
Therapeutic Efficacy of Human Hepatocyte Transplantation in a SCID/uPA Mouse Model with Inducible Liver Disease
Douglas, Donna N.; Kawahara, Toshiyasu; Sis, Banu; Bond, David; Fischer, Karl P.; Tyrrell, D. Lorne J.; Lewis, Jamie T.; Kneteman, Norman M.
Background Severe Combined Immune Deficient (SCID)/Urokinase-type Plasminogen Activator (uPA) mice undergo liver failure and are useful hosts for the propagation of transplanted human hepatocytes (HH) which must compete with recipient-derived hepatocytes for replacement of the diseased liver parenchyma. While partial replacement by HH has proven useful for studies with Hepatitis C virus, complete replacement of SCID/uPA mouse liver by HH has never been achieved and limits the broader application of these mice for other areas of biomedical research. The herpes simplex virus type-1 thymidine kinase (HSVtk)/ganciclovir (GCV) system is a powerful tool for cell-specific ablation in transgenic animals. The aim of this study was to selectively eliminate murine-derived parenchymal liver cells from humanized SCID/uPA mouse liver in order to achieve mice with completely humanized liver parenchyma. Thus, we reproduced the HSVtk (vTK)/GCV system of hepatic failure in SCID/uPA mice. Methodology/Principal Findings In vitro experiments demonstrated efficient killing of vTK expressing hepatoma cells after GCV treatment. For in vivo experiments, expression of vTK was targeted to the livers of FVB/N and SCID/uPA mice. Hepatic sensitivity to GCV was first established in FVB/N mice since these mice do not undergo liver failure inherent to SCID/uPA mice. Hepatic vTK expression was found to be an integral component of GCV-induced pathologic and biochemical alterations and caused death due to liver dysfunction in vTK transgenic FVB/N and non-transplanted SCID/uPA mice. In SCID/uPA mice with humanized liver, vTK/GCV caused death despite extensive replacement of the mouse liver parenchyma with HH (ranging from 32�87%). Surprisingly, vTK/GCV-dependent apoptosis and mitochondrial aberrations were also localized to bystander vTK-negative HH. Conclusions/Significance Extensive replacement of mouse liver parenchyma by HH does not provide a secure therapeutic advantage against vTK/GCV-induced cytotoxicity targeted to residual mouse hepatocytes. Functional support by engrafted HH may be secured by strategies aimed at limiting this bystander effect. PMID:20174638
Genetic Mapping and Functional Studies of a Natural Inhibitor of the Insulin Receptor Tyrosine Kinase: The Mouse Ortholog of Human ?2-HS Glycoprotein
Cintr�n, Vivian J.; Ko, Minoru S. H.; Chi, Kenneth D.; Gross, Jason P.; Srinivas, Pothur R.; Goustin, Anton Scott
Fetuin/?2-HS glycoprotein (?2-HSG) homologs have been identified in several species including rat, sheep, pig, rabbit, guinea pig, cattle, mouse and human. Multiple physiological roles for these homologs have been suggested, including ability to bind to hydroxyapatite crystals and to specifically inhibit the tyrosine kinase (TK) activity of the insulin receptor (IR). In this study we report the identification, cloning, and characterization of the mouse Ahsg gene and its function as an IR-TK inhibitor. Genomic clones derived from a mouse Svj 129 genomic library were sequenced in order to characterize the intron�exon organization of the mouse Ahsg gene, including an 875 bp subclone containing 154 bp upstream from the transcription start site, the first exon, and part of the first intron. A second genomic subclone harboring a 3.45 kb Bgl II fragment contained exons 2, 3 and 4 in addition to two adjacent elements within the first intron-a repetitive element of the B1 family (92 bp) and a 271 bp tract of (T,C)n * (A,G)n. We have mapped mouse Ahsg at 16 cM adjacent to the Diacylglycerol kinase 3 (Dagk3) gene on chromosome 16 by genotyping interspecific backcross panels between C57BL/6J and Mus spretus. The position is syntenic with human chromosome 3q27, where the human AHSG gene resides. Using recombinant mouse ?2-HSG expressed from a recombinant baculovirus, we demonstrate that mouse ?2-HSG inhibits insulin�stimulated IR autophosphorylation and IR-TKA in vitro. In addition, mouse ?2-HSG (25?g/ml) completely abolishes insulin-induced DNA synthesis in H-35 rat hepatoma cells. Based on the sequence data and functional analysis, we conclude that the mouse Ahsg gene is the true ortholog of the human AHSG gene. PMID:11467416
Predictive Dose-Based Estimation of Systemic Exposure Multiples in Mouse and Monkey Relative to Human for Antisense Oligonucleotides With 2?-O-(2-Methoxyethyl) Modifications
Yu, Rosie Z; Grundy, John S; Henry, Scott P; Kim, Tae-Won; Norris, Daniel A; Burkey, Jennifer; Wang, Yanfeng; Vick, Andrew; Geary, Richard S
Evaluation of species differences and systemic exposure multiples (or ratios) in toxicological animal species versus human is an ongoing exercise during the course of drug development. The systemic exposure ratios are best estimated by directly comparing area under the plasma concentration-time curves (AUCs), and sometimes by comparing the dose administered, with the dose being adjusted either by body surface area (BSA) or body weight (BW). In this study, the association between AUC ratio and the administered dose ratio from animals to human were studied using a retrospective data-driven approach. The dataset included nine antisense oligonucleotides (ASOs) with 2?-O-(2-methoxyethyl) modifications, evaluated in two animal species (mouse and monkey) following single and repeated parenteral administrations. We found that plasma AUCs were similar between ASOs within the same species, and are predictable to human exposure using a single animal species, either mouse or monkey. Between monkey and human, the plasma exposure ratio can be predicted directly based on BW-adjusted dose ratios, whereas between mouse and human, the exposure ratio would be nearly fivefold lower in mouse compared to human based on BW-adjusted dose values. Thus, multiplying a factor of 5 for the mouse BW-adjusted dose would likely provide a reasonable AUC exposure estimate in human at steady-state. PMID:25602582
O�Mahony, Orla A.; Steinkamp, Mara P.; Albertelli, Megan A.; Brogley, Michele; Rehman, Haniya; Robins, Diane M.
Gain of function mutations in the androgen receptor (AR) are found in prostate cancer and are implicated in the failure of hormone therapy. Most studies have emphasized the ligand binding domain (LBD) where mutations can create promiscuous receptors, but mutations in the N-terminal transactivation domain (NTD) have also been found. To assess AR alteration as a mechanism of treatment resistance, a mouse model (h/mAR-TRAMP) was used in which the murine AR coding region is replaced by human sequence and prostate cancer initiated by a transgenic oncogene. Mice received either no treatment, androgen-depletion by castration or treatment with antiandrogens, and twenty AR transcripts were sequenced per end-stage tumor. All tumors expressed several mutant alleles, although most mutations were low frequency. Some mutations that occurred multiple times within the population were differentially located dependent on treatment. Mutations in castrated or antiandrogen-treated mice were widely dispersed but with a prominent cluster in the LBD (amino acids 736�771), while changes in intact mice centered near the N-terminal polymorphic glutamine tract. Functional characterization of selected LBD mutant alleles showed diverse effects on AR activity, with about half of the mutations reducing transactivation in vitro. One receptor, AR-R753Q, behaved in a cell- and promoter-dependent manner, even though as a germline mutation it causes androgen insensitivity syndrome. This suggests that alleles that are loss of function during development may still activate a subset of AR targets to become gain of function in tumorigenesis. Mutant ARs may thus utilize multiple mechanisms to evade cancer treatment. PMID:19010817
Imaging the electric field associated with mouse and human skin wounds
Nuccitelli, Richard; Nuccitelli, Pamela; Ramlatchan, Samdeo; Sanger, Richard; Smith, Peter J.S.
We have developed a noninvasive instrument called the bioelectric field imager (BFI) for mapping the electric field between the epidermis and the stratum corneum near wounds in both mouse and human skin. Rather than touching the skin, the BFI vibrates a small metal probe with a displacement of 180 ?m in air above the skin to detect the surface potential of the epidermis through capacitative coupling. Here we describe our first application of the BFI measuring the electric field between the stratum corneum and epidermis at the margin of skin wounds in mice. We measured an electric field of 177 � 14 (61) mV/mm immediately upon wounding and the field lines pointed away from the wound in all directions around it. Because the wound current flows immediately upon wounding, this is the first signal indicating skin damage. This electric field is generated at the outer surface of the epidermis by the outward flow of the current of injury. An equal and opposite current must flow within the multilayered epidermis to generate an intraepidermal field with the negative pole at the wound site. Because the current flowing within the multilayered epidermis is spread over a larger area, the current density and subsequent E field generated in that region is expected to be smaller than that measured by the BFI beneath the stratum corneum. The field beneath the stratum corneum typically remained in the 150�200 mV/mm range for 3 days and then began to decline over the next few days, falling to zero once wound healing was complete. The mean wound field strength decreased by 64 � 7% following the application of the sodium channel blocker, amiloride, to the skin near the wound and increased by 82 � 21% following the application of the Cl� channel activator, prostaglandin E2. PMID:18471262
SIRT1 Is Significantly Elevated in Mouse and Human Prostate Cancer
Derek M. Huffman; William E. Grizzle; Marcas M. Bamman; Jeong-su Kim; Isam A. Eltoum; Ada Elgavish; Tim R. Nagy
Evidence suggests that the histone deacetylase, SIRT1, is a mediator of life span extension by calorie restriction; however, SIRT1 may paradoxically increase the risk of cancer.To better understand the relationship among SIRT1, energy balance, and cancer, two experiments were done.First, a transgenic mouse model of prostate cancer (transgenic adenocarcinoma of mouse prostate; TRAMP) was used to determine the role of
XCI in preimplantation mouse and human embryos: first there is remodelling�
I. M. van den Berg; R. J. Galjaard; J. S. E. Laven; J. H. van Doorninck
Female eutherians silence one of their X chromosomes to accomplish an equal dose of X-linked gene expression compared with\\u000a males. The mouse is the most widely used animal model in XCI research and has proven to be of great significance for understanding\\u000a the complex mechanism of X-linked dosage compensation. Although the basic principles of XCI are similar in mouse and
High-performance liquid chromatography analysis of a novel small-molecule, anti-cancer drug, Palomid 529, in human and mouse plasma and in mouse tissue homogenates.
Lin, Fan; Sherris, David; Beijnen, Jos H; Van Tellingen, Olaf
Palomid 529 (8-(1-Hydroxy-ethyl)-2-methoxy-3-(4-methoxy-benzyloxy)-benzo[c]chromen-6-one), is a novel non-steroidal small-molecule drug, which inhibits both mTORC1 and mTORC2 assembly, and elicits both anti-angiogenic and direct anti-tumor effects in vivo. We have developed and validated a sensitive and selective method for the quantification of Palomid 529 in human and mouse plasma and in a range of mouse tissue samples. Sample pretreatment involved liquid-liquid extraction with tert-butyl methyl ether yielding a recovery of >75%. Palomid 529 and the internal standard Palomid 545 were separated using a GraceSmart RP18 column (2.1 mm � 150 mm) packed with 5 ?m C-18 material and a mobile phase comprised of 50% (v/v) acetonitrile and 50% (v/v) water delivered at a flow rate of 0.2 ml/min, and were detected by UV absorbance at a wavelength of 315 nm. Within the linear range of the calibration curve (10-10,000 ng/ml), acceptable accuracy and precision was achieved for all tested matrices. The validation results show that the method was selective and reproducible. Palomid 529 was stable in plasma upon 3 repeated freeze-thaw cycles and during storage for up to 24h at ambient temperature. However, pre-treated samples waiting for HPLC analyses need to be kept under dimmed light conditions at ambient temperature since a significant degradation of both Palomid 529 and Palomid 545 was observed when exposed to light. A pilot pharmacokinetic study in mice demonstrated the applicability of this method for pharmacokinetic purposes. Even at a low dose of 5.4 mg/kg this assay was still sensitive enough to determine the drug concentration in plasma samples obtained up to 24h after administration. PMID:22100549
Off-target assessment of CRISPR-Cas9 guiding RNAs in human iPS and mouse ES cells.
Tan, E-Pien; Li, Yilong; Velasco-Herrera, Martin Del Castillo; Yusa, Kosuke; Bradley, Allan
The CRISPR-Cas9 system consists of a site-specific, targetable DNA nuclease that holds great potential in gene editing and genome-wide screening applications. To apply the CRISPR-Cas9 system to these assays successfully, the rate at which Cas9 induces DNA breaks at undesired loci must be understood. We characterized the rate of Cas9 off-target activity in typical Cas9 experiments in two human and one mouse cell lines. We analyzed the Cas9 cutting activity of 12 gRNAs in both their targeted sites and ?90 predicted off-target sites per gRNA. In a Cas9-based knockout experiment, gRNAs induced detectable Cas9 cutting activity in all on-target sites and in only a few off-target sites genome-wide in human 293FT, human-induced pluripotent stem (hiPS) cells, and mouse embryonic stem (ES) cells. Both the cutting rates and DNA repair patterns were highly correlated between the two human cell lines in both on-target and off-target sites. In clonal Cas9 cutting analysis in mouse ES cells, biallelic Cas9 cutting was observed with low off-target activity. Our results show that off-target activity of Cas9 is low and predictable by the degree of sequence identity between the gRNA and a potential off-target site. Off-target Cas9 activity can be minimized by selecting gRNAs with few off-target sites of near complementarity. PMID:25378133
Phospholipase C gamma-2 (Plcg2) and phospholipase C gamma-1 (Plcg1) map to distinct regions in the human and mouse genomes.
Argeson, A C; Druck, T; Veronese, M L; Knopf, J L; Buchberg, A M; Huebner, K; Siracusa, L D
The phospholipase C gamma-2 (Plcg2) gene encodes an enzyme that plays a crucial role in intracellular signal transduction pathways. This enzyme is important because of its role in the generation of second messengers following the hydrolysis of phosphatidylinositol 4,5-bisphosphate. We have now determined the chromosomal location of this gene in the mouse and human genomes. An interspecific backcross involving AEJ/Gn and Mus spretus mice was used to localize the gene in mouse. A rodent/human somatic cell hybrid panel was used to map PLCG2 in the human genome. Our results position Plcg2 in the central region of mouse chromosome 8. We also show that PLCG2 maps to the long arm of human chromosome 16, in the region q22-qter. Plcg2 does not map near its most closely related family member, Plcg1, in either genome, indicating that the mammalian Plcg genes belong to a dispersed family. PMID:7774933
Selective destruction of mouse islet beta cells by human T lymphocytes in a newly-established humanized type 1 diabetic model
Zhao, Yong, E-mail: firstname.lastname@example.com [Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612 (United States)] [Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612 (United States); Guo, Chengshan; Hwang, David; Lin, Brian; Dingeldein, Michael; Mihailescu, Dan; Sam, Susan; Sidhwani, Seema [Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612 (United States)] [Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612 (United States); Zhang, Yongkang [Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612 (United States)] [Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Jain, Sumit [Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612 (United States)] [Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612 (United States); Skidgel, Randal A. [Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612 (United States)] [Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Prabhakar, Bellur S. [Department of Immunology and Microbiology, University of Illinois at Chicago, Chicago, IL 60612 (United States)] [Department of Immunology and Microbiology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Mazzone, Theodore [Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612 (United States)] [Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612 (United States); Holterman, Mark J. [Department of Surgery, University of Illinois at Chicago, Chicago, IL 60612 (United States)] [Department of Surgery, University of Illinois at Chicago, Chicago, IL 60612 (United States)
Research highlights: {yields} Establish a human immune-mediated type 1 diabetic model in NOD-scid IL2r{gamma}{sup null} mice. {yields} Using the irradiated diabetic NOD mouse spleen mononuclear cells as trigger. {yields} The islet {beta} cells were selectively destroyed by infiltrated human T cells. {yields} The model can facilitate translational research to find a cure for type 1 diabetes. -- Abstract: Type 1 diabetes (T1D) is caused by a T cell-mediated autoimmune response that leads to the loss of insulin-producing {beta} cells. The optimal preclinical testing of promising therapies would be aided by a humanized immune-mediated T1D model. We develop this model in NOD-scid IL2r{gamma}{sup null} mice. The selective destruction of pancreatic islet {beta} cells was mediated by human T lymphocytes after an initial trigger was supplied by the injection of irradiated spleen mononuclear cells (SMC) from diabetic nonobese diabetic (NOD) mice. This resulted in severe insulitis, a marked loss of total {beta}-cell mass, and other related phenotypes of T1D. The migration of human T cells to pancreatic islets was controlled by the {beta} cell-produced highly conserved chemokine stromal cell-derived factor 1 (SDF-1) and its receptor C-X-C chemokine receptor (CXCR) 4, as demonstrated by in vivo blocking experiments using antibody to CXCR4. The specificity of humanized T cell-mediated immune responses against islet {beta} cells was generated by the local inflammatory microenvironment in pancreatic islets including human CD4{sup +} T cell infiltration and clonal expansion, and the mouse islet {beta}-cell-derived CD1d-mediated human iNKT activation. The selective destruction of mouse islet {beta} cells by a human T cell-mediated immune response in this humanized T1D model can mimic those observed in T1D patients. This model can provide a valuable tool for translational research into T1D.
Comparative mapping of DNA markers from the familial Alzheimer disease and Down syndrome regions of human chromosome 21 to mouse chromosomes 16 and 17
Cheng, S.V.; Nadeau, J.H.; Tanzi, R.E.; Watkins, P.C.; Jagadesh, J.; Taylor, B.A.; Haines, J.L.; Sacchi, N.; Gusella, J.F. (Harvard Medical School, Boston, MA (USA))
Mouse trisomy 16 has been proposed as an animal model of Down syndrome (DS), since this chromosome contains homologues of several loci from the q22 band of human chromosome 21. The recent mapping of the defect causing familial Alzheimer disease (FAD) and the locus encoding the Alzheimer amyloid {beta} precursor protein (APP) to human chromosome 21 has prompted a more detailed examination of the extent of conservation of this linkage group between the two species. Using anonymous DNA probes and cloned genes from human chromosome 21 in a combination of recombinant inbred and interspecific mouse backcross analyses, the authors have established that the linkage group shared by mouse chromosome 16 includes not only the critical DS region of human chromosome 21 but also the APP gene and FAD-linked markers. Extending from the anonymous DNA locus D21S52 to ETS2, the linkage map of six loci spans 39% recombination in man but only 6.4% recombination in the mouse. A break in synteny occurs distal to ETS2, with the homologue of the human marker D21S56 mapping to mouse chromosome 17. Conservation of the linkage relationships of markers in the FAD region suggests that the murine homologue of the FAD locus probably maps to chromosome 16 and that detailed comparison of the corresponding region in both species could facilitate identification of the primary defect in this disorder. The break in synteny between the terminal portion of human chromosome 21 and mouse chromosome 16 indicates, however, that mouse trisomy 16 may not represent a complete model of DS.
Direct activation of human and mouse Oct4 genes using engineered TALE and Cas9 transcription factors
Hu, Jiabiao; Lei, Yong; Wong, Wing-Ki; Liu, Senquan; Lee, Kai-Chuen; He, Xiangjun; You, Wenxing; Zhou, Rui; Guo, Jun-Tao; Chen, Xiongfong; Peng, Xianlu; Sun, Hao; Huang, He; Zhao, Hui; Feng, Bo
The newly developed transcription activator-like effector protein (TALE) and clustered regularly interspaced short palindromic repeats/Cas9 transcription factors (TF) offered a powerful and precise approach for modulating gene expression. In this article, we systematically investigated the potential of these new tools in activating the stringently silenced pluripotency gene Oct4 (Pou5f1) in mouse and human somatic cells. First, with a number of TALEs and sgRNAs targeting various regions in the mouse and human Oct4 promoters, we found that the most efficient TALE-VP64s bound around ?120 to ?80 bp, while highly effective sgRNAs targeted from ?147 to ?89-bp upstream of the transcription start sites to induce high activity of luciferase reporters. In addition, we observed significant transcriptional synergy when multiple TFs were applied simultaneously. Although individual TFs exhibited marginal activity to up-regulate endogenous gene expression, optimized combinations of TALE-VP64s could enhance endogenous Oct4 transcription up to 30-fold in mouse NIH3T3 cells and 20-fold in human HEK293T cells. More importantly, the enhancement of OCT4 transcription ultimately generated OCT4 proteins. Furthermore, examination of different epigenetic modifiers showed that histone acetyltransferase p300 could enhance both TALE-VP64 and sgRNA/dCas9-VP64 induced transcription of endogenous OCT4. Taken together, our study suggested that engineered TALE-TF and dCas9-TF are useful tools for modulating gene expression in mammalian cells. PMID:24500196
Massively Parallel Sequencing Reveals the Complex Structure of an Irradiated Human Chromosome on a Mouse Background in the Tc1 Model of Down Syndrome
Clayton, Stephen; Prigmore, Elena; Langley, Elizabeth; Yang, Fengtang; Maguire, Sean; Fu, Beiyuan; Rajan, Diana; Sheppard, Olivia; Scott, Carol; Hauser, Heidi; Stephens, Philip J.; Stebbings, Lucy A.; Ng, Bee Ling; Fitzgerald, Tomas; Quail, Michael A.; Banerjee, Ruby; Rothkamm, Kai; Tybulewicz, Victor L. J.; Fisher, Elizabeth M. C.; Carter, Nigel P.
Down syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21) and presents a complex phenotype that arises from abnormal dosage of genes on this chromosome. However, the individual dosage-sensitive genes underlying each phenotype remain largely unknown. To help dissect genotype � phenotype correlations in this complex syndrome, the first fully transchromosomic mouse model, the Tc1 mouse, which carries a copy of human chromosome 21 was produced in 2005. The Tc1 strain is trisomic for the majority of genes that cause phenotypes associated with DS, and this freely available mouse strain has become used widely to study DS, the effects of gene dosage abnormalities, and the effect on the basic biology of cells when a mouse carries a freely segregating human chromosome. Tc1 mice were created by a process that included irradiation microcell-mediated chromosome transfer of Hsa21 into recipient mouse embryonic stem cells. Here, the combination of next generation sequencing, array-CGH and fluorescence in situ hybridization technologies has enabled us to identify unsuspected rearrangements of Hsa21 in this mouse model; revealing one deletion, six duplications and more than 25 de novo structural rearrangements. Our study is not only essential for informing functional studies of the Tc1 mouse but also (1) presents for the first time a detailed sequence analysis of the effects of gamma radiation on an entire human chromosome, which gives some mechanistic insight into the effects of radiation damage on DNA, and (2) overcomes specific technical difficulties of assaying a human chromosome on a mouse background where highly conserved sequences may confound the analysis. Sequence data generated in this study is deposited in the ENA database, Study Accession number: ERP000439. PMID:23596509
Localization of a human homolog of the mouse Tiam-1 gene to chromosome 21q22.1
Haiming Chen; Antonarakis, S.E. [Univ. of Geneva Medical School (Switzerland)
Exon trapping was applied to genomic DNA from a chromosome 21-specific cosmid library (LL21NC02-Q) to clone portions of genes from this chromosome. Among a large number of trapped exons, three showed striking homology to different regions of the cDNA for the mouse T-lymphoma invasion and metastasis gene (Tiam-1) at both nucleotide and predicted amino acid sequence levels, suggesting that these three exons are part of a human homolog of the mouse Tiam-1 gene. We mapped this presumed human TIAM1 gene to chromosome 21 by using appropriate somatic cell hybrids, YACs, and cosmids. The TIAM1 gene localizes to YAC 760H5 of the I. Chumakov et al. YAC contig between markers D21S298 and D21S404 in band 21q22.1. This human gene (which is a member of the group of guanine nucleotide-dissociation stimulators that modulate the activity of Rho-like proteins) may be important in the development or metastasis of malignancies that are associated with abnormalities on chromosome 21, including the various forms of leukemia frequent in trisomy 21. 25 refs., 2 figs.
Mouse Mammary Tumor Virus Chromatin in Human Breast Cancer Cells Is Constitutively Hypersensitive and Exhibits Steroid Hormone-Independent Loading of Transcription Factors In Vivo
We have stably introduced a reporter gene under the control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) into human T47D breast cancer cells to study the action of the progesterone receptor (PR) on transcription from a chromatin template. Unexpectedly, the chromatin organization of the MMTV LTR in these human breast cancer cells differed markedly from what
Comparative 32P-analysis of cigarette smoke-induced DNA damage in human tissues and mouse skin.
Randerath, E; Avitts, T A; Reddy, M V; Miller, R H; Everson, R B; Randerath, K
Previous studies using a highly sensitive 32P-postlabeling assay for the analysis of carcinogen/mutagen-induced DNA damage have shown the presence of tobacco smoking-related DNA adducts in human placenta (Everson, R.B., Randerath, E., Santella, R.M., Cefalo, R.C., Avitts, T. A., and Randerath, K., Science (Wash. DC), 231: 54-57, 1986). The occurrence of such adducts in smokers' bronchus and larynx is reported here. Since the chemical nature of these adducts could not be characterized by direct methods due to the extremely low levels of individual adducts (less than 0.03 fmol per microgram DNA), we have sought an experimental animal model for studying the formation of tobacco-related DNA adducts. Because cigarette smoke condensate is known to initiate tumors in mouse skin, ICR mice were treated topically with cigarette tar equivalent to 1.5, 3, 6, and 9 cigarettes for 0.4, 3, 5, and 7 days, respectively, and skin DNA was isolated 1 day after the last treatment. When DNA from exposed mice was analyzed by the 32P-postlabeling assay, 12 distinct 32P-labeled DNA adduct spots, as well as a diagonal radioactive zone, which presumably reflected the presence of incompletely resolved adducts, were noted on polyethyleneimine-cellulose TLC fingerprints. One derivative in particular (adduct 1) was seen to increase rapidly during the early treatment phase and also to persist to 8 days after treatment. The prominent adduct 1 was observed in the same location on the fingerprints of DNA samples from smokers. Cochromatography experiments suggested identity of human and mouse DNA adduct 1. Similarly, several other human and mouse adducts (adducts 3, 5, 6, and 9) appeared identical, and the diagonal radioactive zone was also present on DNA adduct maps from smokers. While absolute levels of individual human adducts were too low to be accurately quantitated, semiquantitative estimation of total tobacco-related aromatic DNA adducts in the human specimens gave values of 1 adduct in (1.7-2.9) X 10(7) nucleotides (0.10-0.18 fmol per micrograms DNA), with adduct 1 constituting 8.5-14% of the total. On the basis of these results, it appears now feasible to determine the chemical origin of smoking-induced DNA adducts in human tissues by preparation of authentic 32P-labeled reference adducts from animals treated with characterized subfractions of cigarette tar, 32P-postlabeling, and cochromatography of 32P-labeled human and animal adducts. PMID:3756927
Antitumor effects of polyethylene glycol-modified recombinant human interleukin-2 on mouse uterine cervical carcinoma in vivo
Lifu Wang; Yuxin Wu; Yongping Zhang; Wei Tang; Xinyuan Liu
Polyethylene glycol (PEG-8000)-modified recombinant human interleukin-2 (PEG-rIL-2) is a cytokine with prolonged circulatory\\u000a half-life. In this paper, the antitumor effects of PEG-rIL-2 against mouse uterine cervical carcinoma (U14) transplanted intraperitoneally or subcutaneously is reported. PEG-rIL-2 at different doses was administered intraperitoneally.\\u000a The results showed that PEG-rIL-2 (4500 IU, i.p., QD�5) prolonged survival time of mice bearing ascites tumor as compared
Mouse Proteomic Technologies Initiative
Mouse models of human cancer offer many opportunities to optimize procedures for profiling major human cancers. The National Cancer Institute's Mouse Proteomic Technologies Initiative, designed to use these animal models to develop and standardize technologies to help improve the accurate measurement of proteins and peptides linked to cancer processes.
Determination of Crizotinib in Human and Mouse Plasma by Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS/MS)
Roberts, Michael S.; Turner, David C.; Broniscer, Alberto; Stewart, Clinton F.
An LC-ESI-MS/MS method using high-throughput solid-phase extraction (SPE) was developed and validated to measure crizotinib in human and mouse plasma to support ongoing clinical and preclinical pharmacokinetic studies. Chromatographic separation of mouse or human plasma extracts was performed on a Supelco Discovery c18 column (50 � 2.1mm, 5.0 �) with gradient elution using a combination of acidified aqueous and methanol (MeOH) mobile phases. The mass-to-charge transition monitored for detection and quantitation of crizotinib was m/z 450.2>260.2 while the stable label internal standard (ISTD) was monitored at m/z 457.2>267.3. The validation studies demonstrated that the assay is both precise and accurate with %CV < 9% and accuracies within 8% of nominal target concentration across all concentrations tested for both the human and mouse plasma matrices. Sample volumes required for analysis were 50 �L and 25 �L for human plasma and mouse plasma, respectively. Calibration curves were linear over a range of 5 � 5000 ng/mL for human plasma and 2 � 2000 ng/mL for mouse plasma. The use of a 96-well plate format enabled rapid extraction as well as compatability with automated workflows. The method was successfully applied to analyze crizotinib concentrations in plasma samples collected from children enrolled on a phase I pediatric study investigating the use of crizotinib for treatment of pediatric brain tumors. PMID:24811158
Human APOE4 increases microglia reactivity at A? plaques in a mouse model of A? deposition
Background Having the apolipoprotein E4 (APOE-?4) allele is the strongest genetic risk factor for the development of Alzheimer�s disease (AD). Accumulation of amyloid beta (A?) in the brain is influenced by APOE genotype. Transgenic mice co-expressing five familial AD mutations (5xFAD) in the presence of human APOE alleles (?2, ?3 or ?4) exhibit APOE genotype-specific differences in early A? accumulation, suggesting an interaction between APOE and AD pathology. Whether APOE genotype affects A?-plaque-associated neuroinflammation remains unclear. In the current study, we address the role of APOE genotype on A?-associated microglial reactivity in the EFAD transgenic mouse model. Methods We analyzed A?-induced glial activation in the brains of 6-month-old EFAD transgenic mice (E2FAD, E3FAD and E4FAD). Region-specific morphological profiles of A? plaques in EFAD brain sections were compared using immunofluorescence staining. We then determined the degree of glial activation in sites of A? deposition while comparing levels of the inflammatory cytokine Interleukin-1? (IL-1?) by ELISA. Finally, we quantified parameters of A?-associated microglial reactivity using double-stained EFAD brain sections. Results Characterization of A? plaques revealed there were larger and more intensely stained plaques in E4FAD mice relative to E2FAD and E3FAD mice. E4FAD mice also had a greater percentage of compact plaques in the subiculum than E3FAD mice. Reactive microglia and dystrophic astrocytes were prominent in EFAD brains, and primarily localized to two sites of significant A? deposition: the subiculum and deep layers of the cortex. Cortical levels of IL-1? were nearly twofold greater in E4FAD mice relative to E3FAD mice. To control for differences in levels of A? in the different EFAD mice, we analyzed the microglia within domains of specific A? deposits. Morphometric analyses revealed increased measures of microglial reactivity in E4FAD mice, including greater dystrophy, increased fluorescence intensity and a higher density of reactive cells surrounding cortical plaques, than in E3FAD mice. Conclusions In addition to altering morphological profiles of A? deposition, APOE genotype influences A?-induced glial activation in the adult EFAD cortex. These data support a role for APOE in modulating A?-induced neuroinflammatory responses in AD progression, and support the use of EFAD mice as a suitable model for mechanistic studies of A?-associated neuroinflammation. PMID:24948358
The SCID-hu Mouse: Murine Model for the Analysis of Human Hematolymphoid Differentiation and Function
J. M. McCune; R. Namikawa; H. Kaneshima; L. D. Shultz; M. Lieberman; I. L. Weissman
The study of human hematopoietic cells and the human immune system is hampered by the lack of a suitable experimental model. Experimental data are presented showing that human fetal liver hematopoietic cells, human fetal thymus, and human fetal lymph node support the differentiation of mature human T cells and B cells after engraftment into mice with genetically determined severe combined
Sequence conservation of the rad21 Schizosaccharomyces pombe DNA double-strand break repair gene in human and mouse
McKay, M.J.; Troelstra, C.; Kanaar, R. [Erasmus Univ., Rotterdam (Netherlands)] [and others] [Erasmus Univ., Rotterdam (Netherlands); and others
The rad21 gene of Schizosaccharomyces pombe is involved in the repair of ionizing radiation-induced DNA double-strand breaks. The isolation of mouse and human putative homologs of rad21 is reported here. Alignment of the predicted amino acid sequence of Rad21 with the mammalian proteins showed that the similarity was distributed across the length of the proteins, with more highly conserved regions at both termini. The mHR21{sup sp} (mouse homolog of Rad21, S. pombe) and hHR21{sup sp} (human homolog of Rad21, S. pombe) predicted proteins were 96% identical, whereas the human and S. pombe proteins were 25% identical and 47% similar. RNA blot analysis showed that mHR21{sup sp} mRNA was abundant in all adult mouse tissues examined, with highest expression in testis and thymus. In addition to a 3.1-kb constitutive mRNA transcript, a 2.2-kb transcript was present at a high level in postmeiotic spermatids, while expression of the 3.1-kb mRNA in testis was confined to the meiotic compartment. hHR21{sup sp} mRNA was cell-cycle regulated in human cells, increasing in late S phase to a peak in G2 phase. The level of hHR21{sup sp} transcripts was not altered by exposure of normal diploid fibroblasts to 10 Gy ionizing radiation. In situ hybridization showed that mHR21{sup sp} resided on chromosome 15D3, whereas hHR21{sup sp} localized to the syntenic 8q24 region. Elevated expression of mHR21{sup sp} in testis and thymus supports a possible role for the rad21 mammalian homologs in V(D)J and meiotic recombination, respectively. Cell cycle regulation of rad21, retained from S. pombe to human, is consistent with a conservation of function between S. pombe and human rad21 genes. 62 refs., 8 figs., 1 tab.
The mouse and human IGSF6 (DORA) genes map to the inflammatory bowel disease 1 locus and are embedded in an intron of a gene of unknown function.
Bates, E E; Kissenpfennig, A; P�ronne, C; Mattei, M G; Fossiez, F; Malissen, B; Lebecque, S
We have previously characterized IGSF6 (DORA), a novel member of the immunoglobulin superfamily (IGSF) from human and rat expressed in dendritic and myeloid cells. Using a probe from the open reading frame of the rat cDNA, we isolated a cosmid which contains the entire mouse gene. By comparative analysis and reverse transcriptase polymerase chain reaction, we defined the intron/exon structure and the mRNA of the mouse gene and, with respect to human BAC clones, the human gene. The genes span 10 kb (mouse) and 12 kb (human), with six exons arranged in a manner similar to other members of the IGSF. All intron/exon boundaries follow the GT-AG rule. Expression of the mouse Igsf6 gene is restricted to cells of the immune system, particularly macrophages. Northern blot revealed a single mRNA of 2.5 kb, in contrast to the human gene which is expressed as two mRNAs of 1 and 2.5 kb. The human and mouse genes were localized to a locus associated with inflammatory bowel disease. Analysis of the flanking regions of the Igsf6 gene revealed the presence of an unrelated gene, transcribed from the opposite strand of the DNA and oriented such that the Igsf6 gene is encoded entirely within an intron. An identical organization is seen in human. This gene of unknown function is transcribed and processed, contains homologues in Caenorhabditis elegans and prokaryotes, and is expressed in most organs in the mouse. PMID:11132146
Staphylococcus aureus alpha-Hemolysin Activates the NLRP3-Inflammasome in Human and Mouse Monocytic Cells
Robin R. Craven; Xi Gao; Irving C. Allen; Denis Gris; Juliane Bubeck Wardenburg; Erin McElvania-Tekippe; Jenny P. Ting; Joseph A. Duncan; Adam J. Ratner
Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA) causes severe necrotizing infections of the skin, soft tissues, and lungs. Staphylococcal ?-hemolysin is an essential virulence factor in mouse models of CA-MRSA necrotizing pneumonia. S. aureus ?-hemolysin has long been known to induce inflammatory signaling and cell death in host organisms, however the mechanism underlying these signaling events were not well understood.
Experimental mouse tumour models: what can be learnt about human cancer immunology?
Glenn Dranoff
The recent demonstration that cancer immunotherapy extends patient survival has reinvigorated interest in elucidating the role of immunity in tumour pathogenesis. Experimental mouse tumour models have provided key mechanistic insights into host antitumour immune responses, and these have guided the development of novel treatment strategies. To accelerate the translation of these findings into clinical benefits, investigators need to gain a
Model organismsThe ascent of mouse: advances in modelling human depression and anxiety
John F. Cryan; Andrew Holmes
Psychiatry has proven to be among the least penetrable clinical disciplines for the development of satisfactory in vivo model systems for evaluating novel treatment approaches. However, mood and anxiety disorders remain poorly understood and inadequately treated. With the explosion in the use of genetically modified mice, enormous research efforts have been focused on developing mouse models of psychiatric disorders. The
Smooth Muscle and Purinergic Contraction of the Human, Rabbit, Rat, and Mouse Testicular Capsule1
Frederick C. L. Banks; Gillian E. Knight; Robert C. Calvert; Mark Turmaine; Cecil S. Thompson; Dimitri P. Mikhailidis; Robert J. Morgan; Geoffrey Burnstock
The smooth-muscle cells of the testicular capsule (tunica albuginea) of man, rat, and mouse were examined by electron microscopy. They were characteristically flattened, elongated, branching cells and diffusely incorporated into the collagenous matrix and did not form a compact muscle layer. Contractile and synthetic smooth-muscle cell phenotypes were identified. Nerve varicosities in close apposition to smooth muscle were seen in
[Effect of combined administration of Angelica polysaccharide and cytarabine on liver of human leukemia NOD/SCID mouse model].
Zhu, Jia-Hong; Xu, Chun-Yan; Mu, Xin-Yi; Liu, Jun; Zhang, Meng-Si; Jia, Dao-Yong; Zhang, Yan-Yan; Huang, Guo-Ning; Wang, Ya-Ping
Leukemia is a type of malignant tumors of hematopoietic system with the abnormal increased immature leukemia cells showing metastasis and invasion ability. Liver is one of the main targets of the leukemia cells spread to, where they may continue to proliferate and differentiate and cause liver function damage, even liver failure. Our previous studies showed that Angelica polysscharides (APS), the main effective components in Angelica sinensis of Chinese traditional medicine, was able to inhibit the proliferation and induced differentiation of the leukemia cells, however, its effect on the liver during the treatment remains elucidated. In the present study, the human leukemia NOD/SCID mouse model were established by implantation human leukemia K562 cells line, then the leukemia mouse were treated with APS, Ara-c or APS + Ara-c respectively by peritoneal injection for 14 days, to explore the effect and mechanism of the chemicals on the mouse liver. Compared to the human leukemia NOD/SCID mouse model group with the treatments of APS, Ara-c and APS + Ara-c, We found that severe liver damage and pathological changes of the liver were able to alleviate: First, the number of white blood cells in the peripheral blood was significantly lower and with less transplanted K562 leukemia cells; Second, liver function damage was alleviated as liver function tests showed that alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBiL) were significantly reduced, while the albumin (Alb) was notably increased; Third, liver antioxidant ability was improved as the activities of the antioxidant enzymes glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were significantly increased, and the contents of GSH and malonaldehyde (MDA) were decreased significantly in the liver; Fourth, the inflammation of the liver was relieved as the level of IL-1beta and IL-6, the inflammatory cytokines, were decreased significantly in the liver. Fifth, liver index was increased as the pathological observation showed that leukemia cells with diffused infiltration into the liver lobules were significantly reduced and with a remarkable increase of apoptotic positive cell rate by TUNEL test. Furthermore, the APS + Ara-c combined administration showed an even more significant positive effect. In conclusion, the APS, Ara-c therapy reduced the accumulation of leukemia cells within the liver, reduced the liver function damage and levels of inflammatory factors, improved antioxidant capacity of the liver tissue and thus alleviate the pathological changes of the liver. Moreover, the APS + Ara-c combination therapy may have an additive effect. PMID:24754180
Sleep Deprivation Impairs Spatial Retrieval but Not Spatial Learning in the Non-Human Primate Grey Mouse Lemur
Rahman, Anisur; Languille, Sol�ne; Lamberty, Yves; Babiloni, Claudio; Perret, Martine; Bordet, Regis; Blin, Olivier J.; Jacob, Tom; Auffret, Alexandra; Schenker, Esther; Richardson, Jill
A bulk of studies in rodents and humans suggest that sleep facilitates different phases of learning and memory process, while sleep deprivation (SD) impairs these processes. Here we tested the hypothesis that SD could alter spatial learning and memory processing in a non-human primate, the grey mouse lemur (Microcebus murinus), which is an interesting model of aging and Alzheimer's disease (AD). Two sets of experiments were performed. In a first set of experiments, we investigated the effects of SD on spatial learning and memory retrieval after one day of training in a circular platform task. Eleven male mouse lemurs aged between 2 to 3 years were tested in three different conditions: without SD as a baseline reference, 8 h of SD before the training and 8 h of SD before the testing. The SD was confirmed by electroencephalographic recordings. Results showed no effect of SD on learning when SD was applied before the training. When the SD was applied before the testing, it induced an increase of the amount of errors and of the latency prior to reach the target. In a second set of experiments, we tested the effect of 8 h of SD on spatial memory retrieval after 3 days of training. Twenty male mouse lemurs aged between 2 to 3 years were tested in this set of experiments. In this condition, the SD did not affect memory retrieval. This is the first study that documents the disruptive effects of the SD on spatial memory retrieval in this primate which may serve as a new validated challenge to investigate the effects of new compounds along physiological and pathological aging. PMID:23717620
Biotin uptake by mouse and human pancreatic beta cells/islets: a regulated, lipopolysaccharide-sensitive carrier-mediated process.
Ghosal, Abhisek; Sekar, Thillai V; Said, Hamid M
Biotin is essential for the normal function of pancreatic beta cells. These cells obtain biotin from their surroundings via transport across their cell membrane. Little is known about the uptake mechanism involved, how it is regulated, and how it is affected by internal and external factors. We addressed these issues using the mouse-derived pancreatic beta-TC-6 cells and freshly isolated mouse and human primary pancreatic beta cells as models. The results showed biotin uptake by pancreatic beta-TC-6 cells occurs via a Na(+)-dependent, carrier-mediated process, that is sensitive to desthiobiotin, as well as to pantothenic acid and lipoate; the process is also saturable as a function of concentration (apparent Km = 22.24 � 5.5 ?M). These cells express the sodium-dependent multivitamin transporter (SMVT), whose knockdown (with doxycycline-inducible shRNA) led to a sever inhibition in biotin uptake. Similarly, uptake of biotin by mouse and human primary pancreatic islets is Na(+)-dependent and carrier-mediated, and both cell types express SMVT. Biotin uptake by pancreatic beta-TC-6 cells is also adaptively regulated (via transcriptional mechanism) by extracellular substrate level. Chronic treatment of pancreatic beta-TC-6 cells with bacterial lipopolysaccharides (LPS) leads to inhibition in biotin uptake. This inhibition is mediated via a Toll-Like receptor 4-mediated process and involves a decrease in membrane expression of SMVT. These findings show, for the first time, that pancreatic beta cells/islets take up biotin via a specific and regulated carrier-mediated process, and that the process is sensitive to the effect of LPS. PMID:24904078
Isolation and characterization of cDNA clones for Humly9: The human homologue of mouse Ly9
Sandrin, M.S.; Henning, M.M.; Lo, M.F. [Austin Research Institute, Heidelberg, Victoria (Australia)] [and others
Ly9 is a mouse cell membrane antigen found on all lymphocytes and coded for by a gene that maps to chromosome 1. We previously described the isolation and characterization of a full-length cDNA clone for mouse Ly9. Using cross-species hybridization we isolated cDNA clones encoding the human homologue Humly9. Analysis of the predicted protein sequence suggests that the extra-cellular portion of the Humly9 molecules is composed of four Ig-like domains: a V domain (V) without disulphide bonds and a truncated C2 domain (tC2) with two disulphide bonds, a second V domain without disulphide bonds and a second tC2 with two disulphide bonds, i.e., as V-tC2-V-tC2. The gene encoding Humly9 was mapped to chromosome 1 by analysis of human/hamster hybrids, and more specifically to the 1q22 region by in situ hybridization. The protein sequence data support the view that Humly9 belongs to the immunoglobulin-superfamily subgroup which includes CD48, CD2, and LFA-3. 20 refs., 5 figs., 1 tab.
p23 co-chaperone protects the aryl hydrocarbon receptor from degradation in mouse and human cell lines.
Nguyen, Phuong Minh; Wang, Depeng; Wang, Yu; Li, Yanjie; Uchizono, James A; Chan, William K
The aryl hydrocarbon receptor (AhR) is a ligand-sensitive transcription factor which is responsible for most 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicities. Without ligand, the AhR complex is cytoplasmic and contains p23. Our objective was to investigate whether the wild type p23 levels are important for the AhR function. We generated eight p23-specific knockdown stable cell lines via either electroporation or lentiviral infection. Five of these stable cell lines were generated from a mouse hepatoma cell line (Hepa1c1c7) and three were from human hepatoma and cervical cell lines (Hep3B and HeLa). All of them expressed lower AhR protein levels, leading to reduced ligand-induced, DRE-driven downstream activity. The AhR protein levels in p23-specific knockdown stable cells were reversed back to wild type levels after exogenous p23 was introduced. Reduction of the AhR protein levels in these stable cells was caused by a decrease in the AhR message levels and an increase of the AhR protein degradation in the absence of ligand. This ligand-independent degradation of AhR was not reversed by MG132, suggesting that the 26S proteasome was not responsible for the degradation. In addition, MG132 could not protect AhR from the ligand-induced degradation in both mouse and human p23-knockdown stable cells. PMID:22759865
SNPs in putative regulatory regions identified by human mouse comparative sequencing and transcription factor binding site data
Banerjee, Poulabi; Bahlo, Melanie; Schwartz, Jody R.; Loots, Gabriela G.; Houston, Kathryn A.; Dubchak, Inna; Speed, Terence P.; Rubin, Edward M.
Genome wide disease association analysis using SNPs is being explored as a method for dissecting complex genetic traits and a vast number of SNPs have been generated for this purpose. As there are cost and throughput limitations of genotyping large numbers of SNPs and statistical issues regarding the large number of dependent tests on the same data set, to make association analysis practical it has been proposed that SNPs should be prioritized based on likely functional importance. The most easily identifiable functional SNPs are coding SNPs (cSNPs) and accordingly cSNPs have been screened in a number of studies. SNPs in gene regulatory sequences embedded in noncoding DNA are another class of SNPs suggested for prioritization due to their predicted quantitative impact on gene expression. The main challenge in evaluating these SNPs, in contrast to cSNPs is a lack of robust algorithms and databases for recognizing regulatory sequences in noncoding DNA. Approaches that have been previously used to delineate noncoding sequences with gene regulatory activity include cross-species sequence comparisons and the search for sequences recognized by transcription factors. We combined these two methods to sift through mouse human genomic sequences to identify putative gene regulatory elements and subsequently localized SNPs within these sequences in a 1 Megabase (Mb) region of human chromosome 5q31, orthologous to mouse chromosome 11 containing the Interleukin cluster.
Statin-induced Kruppel-like factor 2 expression in human and mouse T cells reduces inflammatory and pathogenic responses
Bu, De-xiu; Tarrio, Margarite; Grabie, Nir; Zhang, Yuzhi; Yamazaki, Hiroyuki; Stavrakis, George; Maganto-Garcia, Elena; Pepper-Cunningham, Zachary; Jarolim, Petr; Aikawa, Masanori; Garc�a-Carde�a, Guillermo; Lichtman, Andrew H.
The transcription factor kruppel-like factor 2 (KLF2) is required for the quiescent and migratory properties of naive T cells. Statins, a class of HMG-CoA reductase inhibitors, display pleiotropic immunomodulatory effects that are independent of their lipid-lowering capacity and may be beneficial as therapeutic agents for T cell�mediated inflammatory diseases. Statins upregulate KLF2 expression in endothelial cells, and this activity is associated with an antiinflammatory phenotype. We therefore hypothesized that the immunomodulatory effects of statins are due, in part, to their direct effects on T cell KLF2 gene expression. Here we report that lipophilic statin treatment of mouse and human T cells increased expression of KLF2 through a HMG-CoA/prenylation�dependent pathway. Statins also diminished T cell proliferation and IFN-? expression. shRNA blockade of KLF2 expression in human T cells increased IFN-? expression and prevented statin-induced IFN-? reduction. In a mouse model of myocarditis induced by heart antigen�specific CD8+ T cells, both statin treatment of the T cells and retrovirally mediated overexpression of KLF2 in the T cells had similar ameliorating effects on disease induction. We conclude that statins reduce inflammatory functions and pathogenic activity of T cells through KLF2-dependent mechanisms, and this pathway may be a potential therapeutic target for cardiovascular diseases. PMID:20440076
Metabolism of methylstenbolone studied with human liver microsomes and the uPA?/?-SCID chimeric mouse model.
Geldof, Lore; Lootens, Leen; Polet, Michael; Eichner, Daniel; Campbell, Thane; Nair, Vinod; Botr�, Francesco; Meuleman, Philip; Leroux-Roels, Geert; Deventer, Koen; Eenoo, Peter Van
Anti-doping laboratories need to be aware of evolutions on the steroid market and elucidate steroid metabolism to identify markers of misuse. Owing to ethical considerations, in vivo and in vitro models are preferred to human excretion for nonpharmaceutical grade substances. In this study the chimeric mouse model and human liver microsomes (HLM) were used to elucidate the phase I metabolism of a new steroid product containing, according to the label, methylstenbolone. Analysis revealed the presence of both methylstenbolone and methasterone, a structurally closely related steroid. Via HPLC fraction collection, methylstenbolone was isolated and studied with both models. Using HLM, 10 mono-hydroxylated derivatives (U1-U10) and a still unidentified derivative of methylstenbolone (U13) were detected. In chimeric mouse urine only di-hydroxylated metabolites (U11-U12) were identified. Although closely related, neither methasterone nor its metabolites were detected after administration of isolated methylstenbolone. Administration of the steroid product resulted mainly in the detection of methasterone metabolites, which were similar to those already described in the literature. Methylstenbolone metabolites previously described were not detected. A GC-MS/MS multiple reaction monitoring method was developed to detect methylstenbolone misuse. In one out of three samples, previously tested positive for methasterone, methylstenbolone and U13 were additionally detected, indicating the applicability of the method. PMID:24496964
p23 co-chaperone protects the aryl hydrocarbon receptor from degradation in mouse and human cell lines
Nguyen, Phuong Minh; Wang, Depeng; Wang, Yu; Li, Yanjie; Uchizono, James A.; Chan, William K.
The aryl hydrocarbon receptor (AhR) is a ligand-sensitive transcription factor which is responsible for most 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicities. Without ligand, the AhR complex is cytoplasmic and contains p23. Our objective was to investigate whether the wild type p23 levels are important for the AhR function. We generated eight p23-specific knockdown stable cell lines via either electroporation or lentiviral infection. Five of these stable cell lines were generated from a mouse hepatoma cell line (Hepa1c1c7) and three were from human hepatoma and cervical cell lines (Hep3B and HeLa). All of them expressed lower AhR protein levels, leading to reduced ligand-induced, DRE-driven downstream activity. The AhR protein levels in p23-specific knockdown stable cells were reversed back to wild type levels after exogenous p23 was introduced. Reduction of the AhR protein levels in these stable cells was caused by a decrease in the AhR message levels and an increase of the AhR protein degradation in the absence of ligand. This ligand-independent degradation of AhR was insensitive to MG132, suggesting that the 26S proteasome was not responsible for the degradation. In addition, MG132 could not protect AhR from the ligand-induced degradation in both mouse and human p23-knockdown stable cells. PMID:22759865
Expression of human epidermal growth factor pressures cDNA in transfected mouse NIH 3T3 cells
Mroczkowski, B.; Reich, M.; Whittaker, J.; Bell, G.I.; Cohen, S.
Stable cell lines expressing the human epidermal growth factor (EGF) precursor have been prepared by transfection of mouse NIH 3T3 cells with a bovine papillomavirus-based vector in which the human kidney EGF precursor cDNA has been placed under the control of the inducible mouse metallothionein I promoter. Synthesis of the EGF precursor can be induced by culturing the cells in 5 mM butyric acid or 100 ..mu..M ZnCl/sub 2/. The EGF precursor synthesized by these cells appears to be membrane associated; none is detectable in the cytoplasm. The size of the EGF precursor expressed by these cells is approx. = 150-180 kDa, which is larger than expected from its amino acid sequence, suggesting that it is posttranslationally modified, presumably by glycosylation. The EGF precursor was also detected in the conditioned medium from these cells, indicating that some fraction of the EGF precursor synthesized by these transfected cells may be secreted. Preliminary data suggest that this soluble form of the EGF precursor may compete with /sup 125/I-labeled EGF for binding to the EGF receptor. These cell lines should be useful for studying the processing of the EGF precursor to EGF as well as determining the properties and possible functions of the EGF precursor itself.
Cardiopulmonary dysfunction in the Osteogenesis imperfecta mouse model Aga2 and human patients are caused by bone-independent mechanisms.
Thiele, Frank; Cohrs, Christian M; Flor, Armando; Lisse, Thomas S; Przemeck, Gerhard K H; Horsch, Marion; Schrewe, Anja; Gailus-Durner, Valerie; Ivandic, Boris; Katus, Hugo A; Wurst, Wolfgang; Reisenberg, Catherine; Chaney, Hollis; Fuchs, Helmut; Hans, Wolfgang; Beckers, Johannes; Marini, Joan C; Hrab� de Angelis, Martin
Osteogenesis imperfecta (OI) is an inherited connective tissue disorder with skeletal dysplasia of varying severity, predominantly caused by mutations in the collagen I genes (COL1A1/COL1A2). Extraskeletal findings such as cardiac and pulmonary complications are generally considered to be significant secondary features. Aga2, a murine model for human OI, was systemically analyzed in the German Mouse Clinic by means of in vivo and in vitro examinations of the cardiopulmonary system, to identify novel mechanisms accounting for perinatal lethality. Pulmonary and, especially, cardiac fibroblast of perinatal lethal Aga2/+ animals display a strong down-regulation of Col1a1 transcripts in vivo and in vitro, resulting in a loss of extracellular matrix integrity. In addition, dysregulated gene expression of Nppa, different types of collagen and Agt in heart and lung tissue support a bone-independent vicious cycle of heart dysfunction, including hypertrophy, loss of myocardial matrix integrity, pulmonary hypertension, pneumonia and hypoxia leading to death in Aga2. These murine findings are corroborated by a pediatric OI cohort study, displaying significant progressive decline in pulmonary function and restrictive pulmonary disease independent of scoliosis. Most participants show mild cardiac valvular regurgitation, independent of pulmonary and skeletal findings. Data obtained from human OI patients and the mouse model Aga2 provide novel evidence for primary effects of type I collagen mutations on the heart and lung. The findings will have potential benefits of anticipatory clinical exams and early intervention in OI patients. PMID:22589248
Transcriptional Modulation of Intestinal Innate Defense/Inflammation Genes by Preterm Infant Microbiota in a Humanized Gnotobiotic Mouse Model
Lu, Lei; Yu, Yueyue; Guo, Yuee; Wang, Yunwei; Chang, Eugene B.; Claud, Erika C.
Background and Aims It is known that postnatal functional maturation of the small intestine is facilitated by microbial colonization of the gut. Preterm infants exhibit defects in gut maturation, weak innate immunity against intestinal infection and increased susceptibility to inflammatory disorders, all of which may be related to the inappropriate microbial colonization of their immature intestines. The earliest microbes to colonize the preterm infant gut encounter a na�ve, immature intestine. Thus this earliest microbiota potentially has the greatest opportunity to fundamentally influence intestinal development and immune function. The aim of this study was to characterize the effect of early microbial colonization on global gene expression in the distal small intestine during postnatal gut development. Methods Gnotobiotic mouse models with experimental colonization by early (prior to two weeks of life) intestinal microbiota from preterm human infants were utilized. Microarray analysis was used to assess global gene expression in the intestinal epithelium. Results and Conclusion Multiple intestinal genes involved in metabolism, cell cycle regulation, cell-cell or cell-extracellular matrix communication, and immune function are developmental- and intestinal microbiota- regulated. Using a humanized gnotobiotic mouse model, we demonstrate that certain early preterm infant microbiota from prior to 2 weeks of life specifically induce increased NF-?B activation and a phenotype of increased inflammation whereas other preterm microbiota specifically induce decreased NF-?B activation. These fundamental differences correlate with altered clinical outcomes and suggest the existence of optimal early microbial communities to improve health outcomes. PMID:25928420
Cav1.4 IT mouse as model for vision impairment in human congenital stationary night blindness type 2
Knoflach, Dagmar; Kerov, Vasily; Sartori, Simone B; Obermair, Gerald J; Schmuckermair, Claudia; Liu, Xiaoni; Sothilingam, Vithiyanjali; Garrido, Marina Garcia; Baker, Sheila A; Gl�smann, Martin; Schicker, Klaus; Seeliger, Mathias; Lee, Amy; Koschak, Alexandra
Mutations in the CACNA1F gene encoding the Cav1.4 Ca2+ channel are associated with X-linked congenital stationary night blindness type 2 (CSNB2). Despite the increasing knowledge about the functional behavior of mutated channels in heterologous systems, the pathophysiological mechanisms that result in vision impairment remain to be elucidated. This work provides a thorough functional characterization of the novel IT mouse line that harbors the gain-of-function mutation I745T reported in a New Zealand CSNB2 family.1 Electroretinographic recordings in IT mice permitted a direct comparison with human data. Our data supported the hypothesis that a hyperpolarizing shift in the voltage-dependence of channel activation�as seen in the IT gain-of-function mutant2�may reduce the dynamic range of photoreceptor activity. Morphologically, the retinal outer nuclear layer in adult IT mutants was reduced in size and cone outer segments appeared shorter. The organization of the outer plexiform layer was disrupted, and synaptic structures of photoreceptors had a variable, partly immature, appearance. The associated visual deficiency was substantiated in behavioral paradigms. The IT mouse line serves as a specific model for the functional phenotype of human CSNB2 patients with gain-of-function mutations and may help to further understand the dysfunction in CSNB. PMID:24051672
The viability of mouse spermatogonial germ cells on a novel scaffold, containing human serum albumin and calcium phosphate nanoparticles
Yadegar, Mona; Hekmatimoghaddam, Seyed Hossein; Nezami Saridar, Saeide; Jebali, Ali
Background: In spermatogenesis, spermatogonial cells differentiate to the haploid gametes. It has been shown that spermatogenesis can be done at in vitro condition. In vitro spermatogenesis may provide an open window to treat male infertility. Objective: The aim of this study was to evaluate the effects of a novel scaffold containing human serum albumin (HSA)/tri calcium phosphate nanoparticles (TCP NPs) on the mouse spermatogonial cell line (SCL). Materials and Methods: First, TCP NPs were synthesized by reaction of calcium nitrate and diammonium phosphate at pH 13. Then, serial concentrations of TCP NPs were separately added to 500 mg/mL HSA, and incubated in the 100oC water for 30 min. In the next step, each scaffold was cut (2�2mm), placed into sterile well of microplate, and then incubated for 1, 2, and 3 days at 37oC with mouse SCL. After incubation, the cytotoxicity of the scaffolds was evaluated by different tests including 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) assay, vital staining, and cell counting. On the other hand, the release of TCP NPs and HSA from the scaffolds was measured. Results: Based on microscopic observation, the size of cavities for all scaffolds was near 200-500 �m, and the size of TCP NPs was near 50-100 nm. All toxicity tests showed that the increase of TCP concentration in the scaffold did not affect mouse SCL. It means that the percentage of cell viability, LDH release, vital cells, and cell quantity was 85%, 105%, 90%, and 110%, respectively. But, the increase of incubation time led to increase of LDH release (up to 115%) and cell count (up to 115%). Also, little decrease of cell viability and vital cells was seen when incubation time was increased. Here, no release of TCP NPs and HSA was seen after increase of TCP concentration and incubation time. Conclusion: It can be concluded that the increase of TCP concentration in HSA/ TCP NPs scaffold does not lead to cytotoxicity. On the other hand, the increase of incubation time leads to increase of mouse SCL cell death. In this study, it was found that TCP NPs and HSA could not release from the scaffolds. In future, both proliferation and differentiation of mouse SCL on HSA/TCP NPs scaffold must be checked over more wide incubation times. PMID:26000004
The leukemia inhibitory factor receptor (LIFR) gene is located within a cluster of cytokine receptor loci on mouse chromosome 15 and human chromosome 5p12-p13.
Gearing, D P; Druck, T; Huebner, K; Overhauser, J; Gilbert, D J; Copeland, N G; Jenkins, N A
The leukemia inhibitory factor receptor (LIFR) gene was localized to human chromosome 5p12-p13 by somatic cell hybrid analysis. Interspecific backcross analysis revealed that the murine locus was on chromosome 15 in a region of homology with human chromosome 5p. In both human and mouse genomes, the LIFR locus was linked to the genes encoding the receptors for interleukin-7, prolactin, and growth hormone. PMID:8276403
Influence of Domain Stability on the Properties of Human Apolipoprotein E3 and E4 and Mouse Apolipoprotein E
The human apolipoprotein (apo) E4 isoform, which differs from wild-type apoE3 by the single amino acid substitution C112R, is associated with elevated risk of cardiovascular and Alzheimer�s diseases, but the molecular basis for this variation between isoforms is not understood. Human apoE is a two-domain protein comprising an N-terminal helix bundle and a separately folded C-terminal region. Here, we examine the concept that the ability of the protein to bind to lipid surfaces is influenced by the stability (or readiness to unfold) of these domains. The lipid-free structures and abilities to bind to lipid and lipoprotein particles of a series of human and mouse apoE variants with varying domain stabilities and domain�domain interactions are compared. As assessed by urea denaturation, the two domains are more unstable in apoE4 than in apoE3. To distinguish the contributions of the destabilization of each domain to the greater lipid-binding ability of apoE4, the properties of the apoE4 R61T and E255A variants, which have the same helix bundle stabilities but altered C-terminal domain stabilities, are compared. In these cases, the effects on lipid-binding properties are relatively minor, indicating that the destabilization of the helix bundle domain is primarily responsible for the enhanced lipid-binding ability of apoE4. Unlike human apoE, mouse apoE behaves essentially as a single domain, and its lipid-binding characteristics are more similar to those of apoE4. Together, the results show that the overall stability of the entire apoE molecule exerts a major influence on its lipid- and lipoprotein-binding properties. PMID:24871385
Flaky Tail Mouse Denotes Human Atopic Dermatitis in the Steady State and by Topical Application with Dermatophagoides pteronyssinus Extract
Moniaga, Catharina Sagita; Egawa, Gyohei; Kawasaki, Hiroshi; Hara-Chikuma, Mariko; Honda, Tetsuya; Tanizaki, Hideaki; Nakajima, Saeko; Otsuka, Atsushi; Matsuoka, Hiroyuki; Kubo, Akiharu; Sakabe, Jun-ichi; Tokura, Yoshiki; Miyachi, Yoshiki; Amagai, Masayuki; Kabashima, Kenji
The barrier abnormality, a loss-of-function mutation in the gene encoding filaggrin (FLG), which is linked to the incidence of atopic dermatitis (AD), is a recently discovered but important factor in the pathogenesis of AD. Flaky tail (Flgft) mice, essentially deficient in filaggrin, have been used to investigate the role of filaggrin on AD. However, the relevancy of Flgft mice to human AD needs to be determined further. In this study, we observed the clinical manifestations of Flgft mice in the steady state and their cutaneous immune responses against external stimuli, favoring human AD. Under specific pathogen-free conditions, the majority of Flgft mice developed clinical and histological eczematous skin lesions similar to human AD with outside-to-inside skin barrier dysfunction evaluated by newly devised methods. In addition, cutaneous hapten-induced contact hypersensitivity as a model of acquired immune response and a mite extract-induced dermatitis model physiologically relevant to a human AD were enhanced in Flgft mice. These results suggest that the Flgft mouse genotype has potential as an animal model of AD corresponding with filaggrin mutation in human AD. PMID:20304960
Sequence analysis of the ERCC2 gene regions in human, mouse, and hamster reveals three linked genes
Lamerdin, J.E.; Stilwagen, S.A.; Ramirez, M.H. [Lawrence Livermore National Lab., CA (United States)] [and others] [Lawrence Livermore National Lab., CA (United States); and others
The ERCC2 (excision repair cross-complementing rodent repair group 2) gene product is involved in transcription-coupled repair as an integral member of the basal transcription factor BTF2/TFIIH complex. Defects in this gene can result in three distinct human disorders, namely the cancer-prone syndrome xeroderma pigmentosum complementation group D, trichothiodystrophy, and Cockayne syndrome. We report the comparative analysis of 91.6 kb of new sequence including 54.3 kb encompassing the human ERCC2 locus, the syntenic region in the mouse (32.6 kb), and a further 4.7 kb of sequence 3{prime} of the previously reported ERCC2 region in the hamster. In addition to ERCC2, our analysis revealed the presence of two previously undescribed genes in all three species. The first is centromeric (in the human) to ERCC2 and is most similar to the kinesin light chain gene in sea urchin. The second gene is telomeric (in the human) to ERCC2 and contains a motif found in ankyrins, some cell proteins, and transcription factors. Multiple EST matches to this putative new gene indicate that it is expressed in several human tissues, including breast. The identification and description of two new genes provides potential candidate genes for disorders mapping to this region of 19q13.2. 42 refs., 6 figs., 3 tabs.
Differential expression pattern of ZAC in developing mouse and human pancreas
Xiaoyu Du; Marylene Rousseau; Houria Ounissi-Benkalha; Luc Marchand; Arif Jetha; Steven Paraskevas; Cynthia Goodyer; Constantin Polychronakos
ZAC is a transcription factor and cofactor, a strong candidate for transient neonatal diabetes mellitus (TNDM). TNDM involves\\u000a impaired beta-cell development and is probably due to a double dose of ZAC, which is normally expressed only from the paternal copy. ZAC and Zac1 (its mouse orthologue) are strongly expressed in the proliferating progenitor\\/stem cells in many systems and also in
Staphylococcus aureus ?-Hemolysin Activates the NLRP3-Inflammasome in Human and Mouse Monocytic Cells
Robin R. Craven; Xi Gao; Irving C. Allen; Denis Gris; Juliane Bubeck Wardenburg; Erin McElvania-TeKippe; Jenny P. Ting; Joseph A. Duncan
Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA) causes severe necrotizing infections of the skin, soft tissues, and lungs. Staphylococcal a-hemolysin is an essential virulence factor in mouse models of CA-MRSA necrotizing pneumonia. S. aureus a-hemolysin has long been known to induce inflammatory signaling and cell death in host organisms, however the mechanism underlying these signaling events were not well understood.
Location of mouse and human genes corresponding to conserved canine olfactory receptor gene subfamilies
Ethan A. Carver; Laurie Issel-Tarver; Jasper Rine; Anne S. Olsen; Lisa Stubbs
Olfactory receptors are G protein-coupled, seven-transmembrane-domain proteins that are responsible for binding odorants in\\u000a the nasal epithelium. They are encoded by a large gene family, members of which are organized in several clusters scattered\\u000a throughout the genomes of mammalian species. Here we describe the mapping of mouse sequences corresponding to four conserved\\u000a olfactory receptor genes, each representing separate, recently identified
Caffeine activates mouse TRPA1 channels but suppresses human TRPA1 channels
Katsuhiro Nagatomo; Yoshihiro Kubo
Caffeine has various well-characterized pharmacological effects, but in mammals there are no known plasma membrane receptors or ion channels activated by caffeine. We observed that caffeine activates mouse transient receptor potential A1 (TRPA1) in heterologous expression systems by Cai2+ imaging and electrophysiological analyses. These responses to caffeine were confirmed in acutely dissociated dorsal root ganglion sensory neurons from WT mice,
Seminal vesicles and urinary bladder as sites of aromatization of androgens in men, evidenced by a CYP19A1-driven luciferase reporter mouse and human tissue specimens.
Strauss, Leena; Rantakari, Pia; Sj�gren, Klara; Salminen, Anu; Lauren, Eve; Kallio, Jenny; Damdimopoulou, Pauliina; Bostr�m, Minna; Bostr�m, Peter J; Pakarinen, Pirjo; Zhang, FuPing; Kujala, Paula; Ohlsson, Claes; M�kel�, Sari; Poutanen, Matti
The human CYP19A1 gene is expressed in various tissues by the use of tissue-specific promoters, whereas the rodent cyp19a1 gene is expressed mainly in the gonads and brain. We generated a transgenic mouse model containing a >100-kb 5' region of human CYP19A1 gene connected to a luciferase reporter gene. The luciferase activity in mouse tissues mimicked the CYP19A1 gene expression pattern in humans. Interestingly, the reporter gene activity was 16 and 160 times higher in the urinary bladder and seminal vesicles, respectively, as compared with the activity in the testis. Accordingly, CYP19A1 gene and P450arom protein expression was detected in those human tissues. Moreover, the data revealed that the expression of CYP19A1 gene is driven by promoters PII, I.4, and I.3 in the seminal vesicles, and by promoters PII and I.4 in the urinary bladder. Furthermore, the reporter gene expression in the seminal vesicles was androgen dependent: Castration decreased the expression ?20 times, and testosterone treatment restored it to the level of an intact mouse. This reporter mouse model facilitates studies of tissue-specific regulation of the human CYP19A1 gene, and our data provide evidence for seminal vesicles as important sites for estrogen production in males. PMID:23239821
Biological activity of an intravenous preparation of human vaccinia immune globulin in mouse models of vaccinia virus infection.
Shearer, Jeffry D; Siemann, Linda; Gerkovich, Mary; House, Robert V
The biological activity of a new intravenous (i.v.) preparation of human vaccinia immune globulin (VIGIV) was evaluated in two mouse models of vaccinia virus (VV) infection. In a mouse tail lesion model, female CD-1 mice were inoculated i.v. with 7 x 10(4) PFU of VV to produce >10 lesions per tail 8 days later. In a mouse lethality model, female severe combined immunodeficient (SCID) mice were inoculated i.v. with 3 x 10(4) PFU of VV to produce 100% mortality within 45 days. The ability of VIGIV to reduce tail lesion formation in CD-1 mice and mortality in SCID mice was determined by (i) pretreatment of a lethal VV dose with VIGIV prior to i.v. inoculation into SCID mice and (ii) i.v. administration of VIGIV to CD-1 and SCID mice the day before and up to 8 days after VV infection. VIGIV reduced the proportion of CD-1 mice with >10 tail lesions in a dose-related manner when VIGIV was given 1 day before and up to 1 day after VV inoculation. The pretreatment of VV with VIGIV prolonged survival and decreased mortality. VIGIV (100 and 400 mg/kg) prolonged survival when given up to 4 days after VV inoculation, and the 400-mg/kg dose reduced the mortality rate by 80% when given the day before or immediately after VV inoculation. The biological activity of VIGIV was demonstrated in both the immunocompetent and immunocompromised murine models. The timing of treatment relative to VV inoculation appeared to be important for the demonstration of VIGIV's biological activity. PMID:15980330
Biological Activity of an Intravenous Preparation of Human Vaccinia Immune Globulin in Mouse Models of Vaccinia Virus Infection
Shearer, Jeffry D.; Siemann, Linda; Gerkovich, Mary; House, Robert V.
The biological activity of a new intravenous (i.v.) preparation of human vaccinia immune globulin (VIGIV) was evaluated in two mouse models of vaccinia virus (VV) infection. In a mouse tail lesion model, female CD-1 mice were inoculated i.v. with 7 � 104 PFU of VV to produce >10 lesions per tail 8 days later. In a mouse lethality model, female severe combined immunodeficient (SCID) mice were inoculated i.v. with 3 � 104 PFU of VV to produce 100% mortality within 45 days. The ability of VIGIV to reduce tail lesion formation in CD-1 mice and mortality in SCID mice was determined by (i) pretreatment of a lethal VV dose with VIGIV prior to i.v. inoculation into SCID mice and (ii) i.v. administration of VIGIV to CD-1 and SCID mice the day before and up to 8 days after VV infection. VIGIV reduced the proportion of CD-1 mice with >10 tail lesions in a dose-related manner when VIGIV was given 1 day before and up to 1 day after VV inoculation. The pretreatment of VV with VIGIV prolonged survival and decreased mortality. VIGIV (100 and 400 mg/kg) prolonged survival when given up to 4 days after VV inoculation, and the 400-mg/kg dose reduced the mortality rate by 80% when given the day before or immediately after VV inoculation. The biological activity of VIGIV was demonstrated in both the immunocompetent and immunocompromised murine models. The timing of treatment relative to VV inoculation appeared to be important for the demonstration of VIGIV's biological activity. PMID:15980330
Establishment of a human cell line stably overexpressing mouse Nip45 and characterization of Nip45 subcellular localization
Hashiguchi, Kohtaro; Ozaki, Masumi [Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto (Japan)] [Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto (Japan); Kuraoka, Isao [Biological Chemistry Group, Division of Chemistry, Graduate School of Engineering Science, Osaka University, Osaka (Japan)] [Biological Chemistry Group, Division of Chemistry, Graduate School of Engineering Science, Osaka University, Osaka (Japan); Saitoh, Hisato, E-mail: firstname.lastname@example.com [Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto (Japan) [Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto (Japan); Department of New Frontier Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto (Japan); Global COE (Centers of Excellence) Program, Global Initiative Center for Pulsed Power Engineering, Kumamoto University, Kumamoto (Japan)
Highlights: Black-Right-Pointing-Pointer A human cell line expressing a mouse Nip45 has facilitated Nip45 analysis. Black-Right-Pointing-Pointer Nip45 does not effectively inhibit polySUMOylation in vivo. Black-Right-Pointing-Pointer Nip45 interacts directly with SUMO and SUMO chains. Black-Right-Pointing-Pointer Nip45 accumulates at PML bodies in response to proteasome inhibition. -- Abstract: The nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 2 interacting protein, Nfatc2ip (Nip45), has been implicated as a crucial coordinator of the immune response and of cellular differentiation in humans and mice, and contains SUMO-like domains in its C-terminal region. However, the significance of its N-terminal region and its correlation to the SUMO modification pathway remain largely uncharacterized. In this study, a human cultured cell line was established, in which FLAG-tagged mouse Nip45 (FLAG-mNip45) was stably overexpressed. Under standard, non-stressful conditions, we detected FLAG-mNip45 diffusely distributed in the nucleus. Intriguingly, proteasome inhibition by MG132 caused FLAG-mNip45, together with SUMOylated proteins, to localize in nuclear domains associated with promyelocytic leukemia protein. Finally, using an in vitro binding assay, we showed interaction of the N-terminal region of mNip45 with both free SUMO-3 and SUMO-3 chains, indicating that Nip45 may, in part, exert its function via interaction with SUMO/SUMOylated proteins. Taken together, our study provides novel information on a poorly characterized mammalian protein and suggests that our newly established cell line will be useful for elucidating the physiological role of Nip45.
A Knock-in Mouse Model of Human PHD2 Gene-associated Erythrocytosis Establishes a Haploinsufficiency Mechanism*
Arsenault, Patrick R.; Pei, Fei; Lee, Rebecca; Kerestes, Heddy; Percy, Melanie J.; Keith, Brian; Simon, M. Celeste; Lappin, Terence R. J.; Khurana, Tejvir S.; Lee, Frank S.
The central pathway for controlling red cell mass is the PHD (prolyl hydroxylase domain protein):hypoxia-inducible factor (HIF) pathway. HIF, which is negatively regulated by PHD, activates numerous genes, including ones involved in erythropoiesis, such as the ERYTHROPOIETIN (EPO) gene. Recent studies have implicated PHD2 as the key PHD isoform regulating red cell mass. Studies of humans have identified erythrocytosis-associated, heterozygous point mutations in the PHD2 gene. A key question concerns the mechanism by which human mutations lead to phenotypes. In the present report, we generated and characterized a mouse line in which a P294R knock-in mutation has been introduced into the mouse Phd2 locus to model the first reported human PHD2 mutation (P317R). Phd2P294R/+ mice display a degree of erythrocytosis equivalent to that seen in Phd2+/? mice. The Phd2P294R/+-associated erythrocytosis is reversed in a Hif2a+/?, but not a Hif1a+/? background. Additional studies using various conditional knock-outs of Phd2 reveal that erythrocytosis can be induced by homozygous and heterozygous knock-out of Phd2 in renal cortical interstitial cells using a Pax3-Cre transgene or by homozygous knock-out of Phd2 in hematopoietic progenitors driven by a Vav1-Cre transgene. These studies formally prove that a missense mutation in PHD2 is the cause of the erythrocytosis, show that this occurs through haploinsufficiency, and point to multifactorial control of red cell mass by PHD2. PMID:24121508
A knock-in mouse model of human PHD2 gene-associated erythrocytosis establishes a haploinsufficiency mechanism.
Arsenault, Patrick R; Pei, Fei; Lee, Rebecca; Kerestes, Heddy; Percy, Melanie J; Keith, Brian; Simon, M Celeste; Lappin, Terence R J; Khurana, Tejvir S; Lee, Frank S
The central pathway for controlling red cell mass is the PHD (prolyl hydroxylase domain protein):hypoxia-inducible factor (HIF) pathway. HIF, which is negatively regulated by PHD, activates numerous genes, including ones involved in erythropoiesis, such as the ERYTHROPOIETIN (EPO) gene. Recent studies have implicated PHD2 as the key PHD isoform regulating red cell mass. Studies of humans have identified erythrocytosis-associated, heterozygous point mutations in the PHD2 gene. A key question concerns the mechanism by which human mutations lead to phenotypes. In the present report, we generated and characterized a mouse line in which a P294R knock-in mutation has been introduced into the mouse Phd2 locus to model the first reported human PHD2 mutation (P317R). Phd2(P294R/+) mice display a degree of erythrocytosis equivalent to that seen in Phd2(+/-) mice. The Phd2(P294R/+)-associated erythrocytosis is reversed in a Hif2a(+/-), but not a Hif1a(+/-) background. Additional studies using various conditional knock-outs of Phd2 reveal that erythrocytosis can be induced by homozygous and heterozygous knock-out of Phd2 in renal cortical interstitial cells using a Pax3-Cre transgene or by homozygous knock-out of Phd2 in hematopoietic progenitors driven by a Vav1-Cre transgene. These studies formally prove that a missense mutation in PHD2 is the cause of the erythrocytosis, show that this occurs through haploinsufficiency, and point to multifactorial control of red cell mass by PHD2. PMID:24121508
Expression of UDP-Glucuronosyltransferase 1 (UGT1) and Glucuronidation Activity toward Endogenous Substances in Humanized UGT1 Mouse Brain.
Kutsuno, Yuki; Hirashima, Rika; Sakamoto, Masaya; Ushikubo, Hiroko; Michimae, Hirofumi; Itoh, Tomoo; Tukey, Robert H; Fujiwara, Ryoichi
Although UDP-glucuronosyltransferases (UGTs) are important phase II drug-metabolizing enzymes, they are also involved in the metabolism of endogenous compounds. Certain substrates of UGTs, such as serotonin and estradiol, play important roles in the brain. However, the expression of UGTs in the human brain has not been fully clarified. Recently, humanized UGT1 mice (hUGT1 mice) in which the original Ugt1 locus was disrupted and replaced with the human UGT1 locus have been developed. In the present study, the expression pattern of UGT1As in brains from humans and hUGT1 mice was examined. We found that UGT1A1, 1A3, 1A6, and 1A10 were expressed in human brains. The expression pattern of UGT1As in hUGT1 mouse brains was similar to that in human brains. In addition, we examined the expression of UGT1A1 and 1A6 in the cerebellum, olfactory bulbs, midbrain, hippocampus, and cerebral cortex of hUGT1 mice. UGT1A1 in all brain regions and UGT1A6 in the cerebellum and cerebral cortex of 6-month-old hUGT1 mice were expressed at a significantly higher rate than those of 2-week-old hUGT1 mice. A difference in expression levels between brain regions was also observed. Brain microsomes exhibited glucuronidation activities toward estradiol and serotonin, with mean values of 0.13 and 5.17 pmol/min/mg, respectively. In conclusion, UGT1A1 and UGT1A6 might play an important role in function regulation of endogenous compounds in a region- and age-dependent manner. Humanized UGT1 mice might be useful to study the importance of brain UGTs in vivo. PMID:25953521
S-Adenosylmethionine Regulates Dual-Specificity Mitogen-Activated Protein Kinase Phosphatase Expression in Mouse and Human Hepatocytes
Tomasi, Maria Lauda; Ramani, Komal; Lopitz-Otsoa, Fernando; Rodr�guez, Manuel S.; Li, Tony W. H.; Ko, Kwangsuk; Yang, Heping; Bardag-Gorce, Fawzia; Iglesias-Ara, Ainhoa; Feo, Francesco; Pascale, Maria Rosa; Mato, Jos� M.; Lu, Shelly C.
Increased mitogen-activated protein kinase (MAPK) activity correlates with a more malignant hepatocellular carcinoma (HCC) phenotype. There is a reciprocal regulation between p44/42 MAPK (extracellular signal-regulated kinase [ERK]1/2) and the dual-specificity MAPK phosphatase MKP-1/DUSP1. ERK phosphorylates DUSP1, facilitating its proteasomal degradation, whereas DUSP1 inhibits ERK activity. Methionine adenosyltransferase 1a (Mat1a) knockout (KO) mice express hepatic S-adenosylmethionine (SAM) deficiency and increased ERK activity and develop HCC. The aim of this study was to examine whether DUSP1 expression is regulated by SAM and if so, elucidate the molecular mechanisms. Studies were conducted using Mat1a KO mice livers, cultured mouse and human hepatocytes, and 20S and 26S proteasomes. DUSP1 messenger RNA (mRNA) and protein levels were reduced markedly in livers of Mat1a KO mice and in cultured mouse and human hepatocytes with protein falling to lower levels than mRNA. SAM treatment protected against the fall in DUSP1 mRNA and protein levels in mouse and human hepatocytes. SAM increased DUSP1 transcription, p53 binding to DUSP1 promoter, and stability of its mRNA and protein. Proteasomal chymotrypsin-like and caspase-like activities were increased in Mat1a KO livers and cultured hepatocytes, which was blocked by SAM treatment. SAM inhibited chymotrypsin-like and caspase-like activities by 40% and 70%, respectively, in 20S proteasomes and caused rapid degradation of some of the 26S proteasomal subunits, which was blocked by the proteasome inhibitor MG132. SAM treatment in Mat1a KO mice for 7 days raised SAM, DUSP1, mRNA and protein levels and lowered proteosomal and ERK activities. Conclusion DUSP1 mRNA and protein levels are lower in Mat1a KO livers and fall rapidly in cultured hepatocytes. SAM treatment increases DUSP1 expression through multiple mechanisms, and this may suppress ERK activity and malignant degeneration. PMID:20196119
Genome-Wide and Species-Wide In Silico Screening for Intragenic MicroRNAs in Human, Mouse and Chicken
Godnic, Irena; Zorc, Minja; Jevsinek Skok, Dasa; Calin, George Adrian; Horvat, Simon; Dovc, Peter; Kovac, Milena; Kunej, Tanja
MicroRNAs (miRNAs) are non-coding RNAs (ncRNAs) involved in regulation of gene expression. Intragenic miRNAs, especially those exhibiting a high degree of evolutionary conservation, have been shown to be coordinately regulated and/or expressed with their host genes, either with synergistic or antagonistic correlation patterns. However, the degree of cross-species conservation of miRNA/host gene co-location is not known and co-expression information is incomplete and fragmented among several studies. Using the genomic resources (miRBase and Ensembl) we performed a genome-wide in silico screening (GWISS) for miRNA/host gene pairs in three well-annotated vertebrate species: human, mouse, and chicken. Approximately half of currently annotated miRNA genes resided within host genes: 53.0% (849/1,600) in human, 48.8% (418/855) in mouse, and 42.0% (210/499) in chicken, which we present in a central publicly available Catalog of intragenic miRNAs (http://www.integratomics-time.com/miR-host/catalog). The miRNA genes resided within either protein-coding or ncRNA genes, which include long intergenic ncRNAs (lincRNAs) and small nucleolar RNAs (snoRNAs). Twenty-seven miRNA genes were found to be located within the same host genes in all three species and the data integration from literature and databases showed that most (26/27) have been found to be co-expressed. Particularly interesting are miRNA genes located within genes encoding for miRNA silencing machinery (DGCR8, DICER1, and SND1 in human and Cnot3, Gdcr8, Eif4e, Tnrc6b, and Xpo5 in mouse). We furthermore discuss a potential for phenotype misattribution of miRNA host gene polymorphism or gene modification studies due to possible collateral effects on miRNAs hosted within them. In conclusion, the catalog of intragenic miRNAs and identified 27 miRNA/host gene pairs with cross-species conserved co-location, co-expression, and potential co-regulation, provide excellent candidates for further functional annotation of intragenic miRNAs in health and disease. PMID:23762306
A Mouse Model of Human Hyperinsulinism Produced by the E1506K Mutation in the Sulphonylurea Receptor SUR1
Shimomura, Kenju; Tusa, Maija; Iberl, Michaela; Brereton, Melissa F.; Kaizik, Stephan; Proks, Peter; Lahmann, Carolina; Yaluri, Nagendra; Modi, Shalem; Huopio, Hanna; Ustinov, Jarkko; Otonkoski, Timo; Laakso, Markku; Ashcroft, Frances M.
Loss-of-function mutations in the KATP channel genes KCNJ11 and ABCC8 cause neonatal hyperinsulinism in humans. Dominantly inherited mutations cause less severe disease, which may progress to glucose intolerance and diabetes in later life (e.g., SUR1-E1506K). We generated a mouse expressing SUR1-E1506K in place of SUR1. KATP channel inhibition by MgATP was enhanced in both homozygous (homE1506K) and heterozygous (hetE1506K) mutant mice, due to impaired channel activation by MgADP. As a consequence, mutant ?-cells showed less on-cell KATP channel activity and fired action potentials in glucose-free solution. HomE1506K mice exhibited enhanced insulin secretion and lower fasting blood glucose within 8 weeks of birth, but reduced insulin secretion and impaired glucose tolerance at 6 months of age. These changes correlated with a lower insulin content; unlike wild-type or hetE1506K mice, insulin content did not increase with age in homE1506K mice. There was no difference in the number and size of islets or ?-cells in the three types of mice, or evidence of ?-cell proliferation. We conclude that the gradual development of glucose intolerance in patients with the SUR1-E1506K mutation might, as in the mouse model, result from impaired insulin secretion due a failure of insulin content to increase with age. PMID:23903354
In Vivo Imaging of Human and Mouse Skin with a Handheld Dual-Axis Confocal Fluorescence Microscope
Gonzalez-Gonzalez, Emilio; Mandella, Michael J.; Kino, Gordon S.; Solgaard, Olav; Leake, Devin; Kaspar, Roger L.; Oro, Anthony; Contag, Christopher H.
Advancing molecular therapies for the treatment of skin diseases will require the development of new tools that can reveal spatiotemporal changes in the microanatomy of the skin and associate these changes with the presence of the therapeutic agent. For this purpose, we evaluated a handheld dual-axis confocal (DAC) microscope that is capable of in vivo fluorescence imaging of skin, using both mouse models and human skin. Individual keratinocytes in the epidermis were observed in three-dimensional image stacks after topical administration of near-infrared (NIR) dyes as contrast agents. This suggested that the DAC microscope may have utility in assessing the clinical effects of a small interfering RNA (siRNA)-based therapeutic (TD101) that targets the causative mutation in pachyonychia congenita (PC) patients. The data indicated that (1) formulated indocyanine green (ICG) readily penetrated hyperkeratotic PC skin and normal callused regions compared with nonaffected areas, and (2) TD101-treated PC skin revealed changes in tissue morphology, consistent with reversion to nonaffected skin compared with vehicle-treated skin. In addition, siRNA was conjugated to NIR dye and shown to penetrate through the stratum corneum barrier when topically applied to mouse skin. These results suggest that in vivo confocal microscopy may provide an informative clinical end point to evaluate the efficacy of experimental molecular therapeutics. PMID:21191407
YAP regulates the expression of Hoxa1 and Hoxc13 in mouse and human oral and skin epithelial tissues.
Liu, Ming; Zhao, Shuangyun; Lin, Qingjie; Wang, Xiu-Ping
Yes-associated protein (YAP) is a Hippo signaling transcriptional coactivator that plays pivotal roles in stem cell proliferation, organ size control, and tumor development. The downstream targets of YAP have been shown to be highly context dependent. In this study, we used the embryonic mouse tooth germ as a tool to search for the downstream targets of YAP in ectoderm-derived tissues. Yap deficiency in the dental epithelium resulted in a small tooth germ with reduced epithelial cell proliferation. We compared the gene expression profiles of embryonic day 14.5 (E14.5) Yap conditional knockout and YAP transgenic mouse tooth germs using transcriptome sequencing (RNA-Seq) and further confirmed the differentially expressed genes using real-time PCR and in situ hybridization. We found that YAP regulates the expression of Hoxa1 and Hoxc13 in oral and dental epithelial tissues as well as in the epidermis of skin during embryonic and adult stages. Sphere formation assay suggested that Hoxa1 and Hoxc13 are functionally involved in YAP-regulated epithelial progenitor cell proliferation, and chromatin immunoprecipitation (ChIP) assay implies that YAP may regulate Hoxa1 and Hoxc13 expression through TEAD transcription factors. These results provide mechanistic insights into abnormal YAP activities in mice and humans. PMID:25691658
Isolation of Anaerobic Bacteria from Human Gingiva and Mouse Cecum by Means of a Simplified Glove Box Procedure1
Aranki, Alexander; Syed, Salam A.; Kenney, Ernest B.; Freter, Rolf
An anaerobic glove box constructed of clear flexible vinyl plastic is described. It is sufficiently inexpensive and simple in operation to be used not only in research but also in a clinical laboratory by technicians without special training. Conventional bacteriological techniques may be used inside the glove box for culturing and transferring anaerobic bacteria. The box may be heated to 37 C and thus serve as an anaerobic incubator as well, permitting inspection of cultures at any time. Media may be prepared and agar plates may be poured on the laboratory bench in the conventional manner. An overlay of trace amounts of palladium black catalyst over plated agar media reduces the medium to an oxidation-reduction (O-R) potential of - 300 mv within 2 days after introduction into the glove box. In spite of its greater simplicity, the system matched or excelled the roll tube method with respect to all parameters tested, including O-R potential obtainable in the media, O2 concentration in the gas phase, and efficiency in isolating anaerobic bacteria from the mouse cecum. Comparative studies indicate that the conventional anaerobic jar method was inadequate for the isolation of strict anaerobes from human gingival specimens and from the mouse cecum. This was due to the exposure of specimens and media to air during plating on the open laboratory bench. Anaerobic jars were adequate for maintaining the proper conditions for growth of anaerobic bacteria once these had been established in the glove box. Images PMID:4890748
Retroviral transduction of the human c-Ha-ras-1 oncogene into midgestation mouse embryos promotes rapid epithelial hyperplasia
Compere, S.J.; Baldacci, P.A.; Jaenisch, R.
Infection of mouse embryos at 8 days of gestation with a replication-defective retrovirus carrying the human c-Ha-ras-1 oncogene led to efficient and rapid induction of hyperplastic lesions. Twenty-four percent of viable offspring developed abnormal growths after infection with purified virus. The lesions contained a single integrated provirus and produced viral RNA and the Ha-ras oncogene product (p21). The latency period between the time of infection and appearance of the lesions suggested that secondary alterations in addition to activated ras were necessary for neoplasms to develop. The earliest and most abundant growths were cutaneous and appeared from 4 to 36 weeks of age, with a median of 4 weeks of age. A number of subcutaneous lesions also developed over the same time span but at a median of 18 weeks of age. The rapid development of cutaneous lesions in response to transduction of the ras oncogene contrasts with other studies in which adult skin required secondary treatment with promoters prior to ras induction of epithelial hyperplasia. These results demonstrate that infection of midgestation mouse embryos allows rapid analysis of oncogene potency in skin.
Erythrocytosis and Pulmonary Hypertension in a Mouse Model of Human HIF2A Gain of Function Mutation*
Tan, Qiulin; Kerestes, Heddy; Percy, Melanie J.; Pietrofesa, Ralph; Chen, Li; Khurana, Tejvir S.; Christofidou-Solomidou, Melpo; Lappin, Terence R. J.; Lee, Frank S.
The central pathway for oxygen-dependent control of red cell mass is the prolyl hydroxylase domain protein (PHD):hypoxia inducible factor (HIF) pathway. PHD site specifically prolyl hydroxylates the transcription factor HIF-?, thereby targeting the latter for degradation. Under hypoxia, this modification is attenuated, allowing stabilized HIF-? to activate target genes, including that for erythropoietin (EPO). Studies employing genetically modified mice point to Hif-2?, one of two main Hif-? isoforms, as being the critical regulator of Epo in the adult mouse. More recently, erythrocytosis patients with heterozygous point mutations in the HIF2A gene have been identified; whether these mutations were polymorphisms unrelated to the phenotype could not be ruled out. In the present report, we characterize a mouse line bearing a G536W missense mutation in the Hif2a gene that corresponds to the first such human mutation identified (G537W). We obtained mice bearing both heterozygous and homozygous mutations at this locus. We find that these mice display, in a mutation dose-dependent manner, erythrocytosis and pulmonary hypertension with a high degree of penetrance. These findings firmly establish missense mutations in HIF-2? as a cause of erythrocytosis, highlight the importance of this HIF-? isoform in erythropoiesis, and point to physiologic consequences of HIF-2? dysregulation. PMID:23640890
A novel mouse model of endometriosis mimics human phenotype and reveals insights into the inflammatory contribution of shed endometrium.
Greaves, Erin; Cousins, Fiona L; Murray, Alison; Esnal-Zufiaurre, Arantza; Fassbender, Amelie; Horne, Andrew W; Saunders, Philippa T K
Endometriosis is an estrogen-dependent inflammatory disorder characterized by the presence of endometrial tissue outside the uterine cavity. Patients experience chronic pelvic pain and infertility, with the most likely origin of the tissue deposits (lesions) being endometrial fragments shed at menses. Menstruation is an inflammatory process associated with a dramatic increase in inflammatory mediators and tissue-resident immune cells. In the present study, we developed and validated a mouse model of endometriosis using syngeneic menstrual endometrial tissue introduced into the peritoneum of immunocompetent mice. We demonstrate the establishment of endometriotic lesions that exhibit similarities to those recovered from patients undergoing laparoscopy. Specifically, in both cases, lesions had epithelial (cytokeratin(+)) and stromal (vimentin/CD10(+)) cell compartments with a well-developed vasculature (CD31(+) endothelial cells). Expression of estrogen receptor ? was increased in lesions compared with the peritoneum or eutopic endometrium. By performing experiments using mice with green fluorescent protein-labeled macrophages (MacGreen) in reciprocal transfers with wild-type mice, we obtained evidence that macrophages present in the peritoneum and in menses endometrium can contribute to the inflammatory microenvironment of the lesions. In summary, we developed a mouse model of endometriosis that exhibits similarities to human peritoneal lesions with respect to estrogen receptor expression, inflammation, and macrophage infiltration, providing an opportunity for further studies and the possible identification of novel therapies for this perplexing disorder. PMID:24910298
A mutation in Tubb2b, a human polymicrogyria gene, leads to lethality and abnormal cortical development in the mouse
Stottmann, R.W.; Donlin, M.; Hafner, A.; Bernard, A.; Sinclair, D.A.; Beier, D.R.
Human cortical malformations, including lissencephaly, polymicrogyria and other diseases of neurodevelopment, have been associated with mutations in microtubule subunits and microtubule-associated proteins. Here we report our cloning of the brain dimple (brdp) mouse mutation, which we recovered from an ENU screen for recessive perinatal phenotypes affecting neurodevelopment. We identify the causal mutation in the tubulin, beta-2b (Tubb2b) gene as a missense mutation at a highly conserved residue (N247S). Brdp/brdp homozygous mutants have significant thinning of the cortical epithelium, which is markedly more severe in the caudo-lateral portion of the telencephalon, and do not survive past birth. The cortical defects are largely due to a major increase in apoptosis and we note abnormal proliferation of the basal progenitors. Adult brdp/+ mice are viable and fertile but exhibit behavioral phenotypes. This allele of Tubb2b represents the most severely affected mouse tubulin phenotype reported to date and this is the first report of a tubulin mutation affecting neuronal proliferation and survival. PMID:23727838
A Nude Mouse Model for Human Bone Formation in Unloaded Conditions
A Muraglia; I Martin; R Cancedda; R Quarto
We describe an experimental model for human bone formation in unloaded conditions. Bone formation has been assessed by implanting in vivo human bone marrow stromal cells (BMSC) on porous hydroxyapatite (HA) bioceramics subcutaneously in nude mice. In this system, human bone formation and remodeling occurs and can be studied in unloaded conditions, i.e., with no influence of muscle tension. Using
Targeting Tumor Vasculature Endothelial Cells and Tumor Cells for Immunotherapy of Human Melanoma in a Mouse Xenograft Model
NASA Astrophysics Data System (ADS)
Hu, Zhiwei; Sun, Ying; Garen, Alan
An immunotherapy treatment for cancer that targets both the tumor vasculature and tumor cells has shown promising results in a severe combined immunodeficient mouse xenograft model of human melanoma. The treatment involves systemic delivery of an immunoconjugate molecule composed of a tumor-targeting domain conjugated to the Fc effector domain of human IgG1. The effector domain induces a cytolytic immune response against the targeted cells by natural killer cells and complement. Two types of targeting domains were used. One targeting domain is a human single-chain Fv molecule that binds to a chondroitin sulfate proteoglycan expressed on the surface of most human melanoma cells. Another targeting domain is factor VII (fVII), a zymogen that binds with high specificity and affinity to the transmembrane receptor tissue factor (TF) to initiate the blood coagulation cascade. TF is expressed by endothelial cells lining the tumor vasculature but not the normal vasculature, and also by many types of tumor cells including melanoma. Because the binding of a fVII immunoconjugate to TF might cause disseminated intravascular coagulation, the active site of fVII was mutated to inhibit coagulation without affecting the affinity for TF. The immunoconjugates were encoded as secreted molecules in a replication-defective adenovirus vector, which was injected into the tail vein of severe combined immunodeficient mice. The results demonstrate that a mutated fVII immunoconjugate, administered separately or together with a single-chain Fv immunoconjugate that binds to the tumor cells, can inhibit the growth or cause regression of an established human tumor xenograft. This procedure could be effective in treating a broad spectrum of human solid tumors that express TF on vascular endothelial cells and tumor cells.
Herpes simplex virus 1 tropism for human sensory ganglion neurons in the severe combined immunodeficiency mouse model of neuropathogenesis.
Zerboni, Leigh; Che, Xibing; Reichelt, Mike; Qiao, Yanli; Gu, Haidong; Arvin, Ann
The tropism of herpes simplex virus (HSV-1) for human sensory neurons infected in vivo was examined using dorsal root ganglion (DRG) xenografts maintained in mice with severe combined immunodeficiency (SCID). In contrast to the HSV-1 lytic infectious cycle in vitro, replication of the HSV-1 F strain was restricted in human DRG neurons despite the absence of adaptive immune responses in SCID mice, allowing the establishment of neuronal latency. At 12 days after DRG inoculation, 26.2% of human neurons expressed HSV-1 protein and 13.1% expressed latency-associated transcripts (LAT). Some infected neurons showed cytopathic changes, but HSV-1, unlike varicella-zoster virus (VZV), only rarely infected satellite cells and did not induce fusion of neuronal and satellite cell plasma membranes. Cell-free enveloped HSV-1 virions were observed, indicating productive infection. A recombinant HSV-1-expressing luciferase exhibited less virulence than HSV-1 F in the SCID mouse host, enabling analysis of infection in human DRG xenografts for a 61-day interval. At 12 days after inoculation, 4.2% of neurons expressed HSV-1 proteins; frequencies increased to 32.1% at 33 days but declined to 20.8% by 61 days. Frequencies of LAT-positive neurons were 1.2% at 12 days and increased to 40.2% at 33 days. LAT expression remained at 37% at 61 days, in contrast to the decline in neurons expressing viral proteins. These observations show that the progression of HSV-1 infection is highly restricted in human DRG, and HSV-1 genome silencing occurs in human neurons infected in vivo as a consequence of virus-host cell interactions and does not require adaptive immune control. PMID:23269807
Among the nitro-substituted polycyclic aromatic hydrocarbons identified in environmental samples and known to be genotoxic, 1-nitropyrene is one of the most abundant. he biotransformation of 1-nitro[14C]pyrene by human, rat, and mouse intestinal microflora and the mutagenicity of...
A xenograft mouse model coupled with in-depth plasma proteome analysis facilitates identification of novel serum biomarkers for human ovarian cancer
Tang, Hsin-Yao; Beer, Lynn A.; Chang-Wong, Tony; Hammond, Rachel; Gimotty, Phyllis; Coukos, George; Speicher, David W.
Proteomics discovery of novel cancer serum biomarkers is hindered by the great complexity of serum, patient-to-patient variability, and triggering by the tumor of an acute-phase inflammatory reaction. This host response alters many serum protein levels in cancer patients, but these changes have low specificity as they can be triggered by diverse causes. We addressed these hurdles by utilizing a xenograft mouse model coupled with an in-depth 4-D protein profiling method to identify human proteins in the mouse serum. This strategy ensures identified putative biomarkers are shed by the tumor, and detection of low-abundance proteins shed by the tumor is enhanced because the mouse blood volume is more than a thousand times smaller than that of a human. Using TOV-112D ovarian tumors, more than 200 human proteins were identified in the mouse serum, including novel candidate biomarkers and proteins previously reported to be elevated in either ovarian tumors or the blood of ovarian cancer patients. Subsequent quantitation of selected putative biomarkers in human sera using label-free multiple reaction monitoring (MRM) mass spectrometry (MS) showed that chloride intracellular channel 1, the mature form of cathepsin D, and peroxiredoxin 6 were elevated significantly in sera from ovarian carcinoma patients. PMID:22032327
DNA adducts in human and mouse skin maintained in short-term culture and treated with petrol and diesel engine lubricating oils.
Carmichael, P L; Ni Sh�, M; Phillips, D H
Human and mouse skin samples maintained in short-term organ culture were treated topically with used engine oils from petrol- and diesel-powered vehicles. Mice were also treated topically in vivo for comparison. DNA was isolated and analysed by 32P-postlabelling and the labeled DNA digests were resolved on polyethyleneimine-cellulose tlc sheets. A large number of radioactive adduct spots were observed in DNA from skin treated with the used petrol-engine oil, indicating the formation of adducts by many components of the complex oil mixture. Total adduct levels were similar in mouse skin (both in vivo and in vitro) and in human skin, although qualitative differences in the adduct maps were apparent between the human and mouse skin DNA. Treatment with the used diesel engine oil produced adduct levels no greater than that of control samples in mouse skin (in vivo and in vitro), although significant levels were found in human skin DNA from one donor. The results correlate well with the carcinogenic activity of these oils in experimental animals, helping to substantiate the conclusion that petrol engine oils (but not diesel engine oils) may present a carcinogenic risk to man if appropriate measures to minimise skin contact are not observed. PMID:2032211
Genetic basis of neural tube defects: the mouse gene loop-tail maps to a region of chromosome 1 syntenic with human 1q21�q23
Philip Stanier; Jennifer N. Henson; Jane Eddleston; Gudrun E. Moore; Andrew J. Copp
A genetic basis for neural tube defects (NTD) is rarely doubted, but the genes involved have not yet been identified. This is partly due to a lack of suitable families on which to perform linkage analysis. An alternative approach is to use the many mouse genes that cause NTD as a means of isolating their human homologues. Loop-tail (Lp) is
Bone Marrow Transplantation Results in Human Donor Blood Cells Acquiring and Displaying Mouse Recipient Class I MHC and CD45 Antigens on Their Surface
Nobuko Yamanaka; Christine J. Wong; Marina Gertsenstein; Robert F. Casper; Andras Nagy; Ian M. Rogers; Joseph Najbauer
BackgroundMouse models of human disease are invaluable for determining the differentiation ability and functional capacity of stem cells. The best example is bone marrow transplants for studies of hematopoietic stem cells. For organ studies, the interpretation of the data can be difficult as transdifferentiation, cell fusion or surface antigen transfer (trogocytosis) can be misinterpreted as differentiation. These events have not
Eukaryotic expression, purification and structure/function analysis of native, recombinant CRISP3 from human and mouse
NASA Astrophysics Data System (ADS)
Volpert, Marianna; Mangum, Jonathan E.; Jamsai, Duangporn; D'Sylva, Rebecca; O'Bryan, Moira K.; McIntyre, Peter
While the Cysteine-Rich Secretory Proteins (CRISPs) have been broadly proposed as regulators of reproduction and immunity, physiological roles have yet to be established for individual members of this family. Past efforts to investigate their functions have been limited by the difficulty of purifying correctly folded CRISPs from bacterial expression systems, which yield low quantities of correctly folded protein containing the eight disulfide bonds that define the CRISP family. Here we report the expression and purification of native, glycosylated CRISP3 from human and mouse, expressed in HEK 293 cells and isolated using ion exchange and size exclusion chromatography. Functional authenticity was verified by substrate-affinity, native glycosylation characteristics and quaternary structure (monomer in solution). Validated protein was used in comparative structure/function studies to characterise sites and patterns of N-glycosylation in CRISP3, revealing interesting inter-species differences.
The forkhead transcription factor Foxl2 is sumoylated in both human and mouse: sumoylation affects its stability, localization, and activity.
Marongiu, Mara; Deiana, Manila; Meloni, Alessandra; Marcia, Loredana; Puddu, Alessandro; Cao, Antonio; Schlessinger, David; Crisponi, Laura
The FOXL2 forkhead transcription factor is expressed in ovarian granulosa cells, and mutated FOXL2 causes the blepharophimosis, ptosis and epicanthus inversus syndrome (BPES) and predisposes to premature ovarian failure. Inactivation of Foxl2 in mice demonstrated its indispensability for female gonadal sex determination and ovary development and revealed its antagonism of Sox9, the effector of male testis development. To help to define the regulatory activities of FOXL2, we looked for interacting proteins. Based on yeast two-hybrid screening, we found that FOXL2 interacts with PIAS1 and UBC9, both parts of the sumoylation machinery. We showed that human FOXL2 is sumoylated in transfected cell lines, and that endogenous mouse Foxl2 is comparably sumoylated. This modification changes its cellular localization, stability and transcriptional activity. It is intriguing that similar sumoylation and regulatory consequences have also been reported for SOX9, the male counterpart of FOXL2 in somatic gonadal tissues. PMID:20209145
The Forkhead Transcription Factor Foxl2 Is Sumoylated in Both Human and Mouse: Sumoylation Affects Its Stability, Localization, and Activity
Marongiu, Mara; Deiana, Manila; Meloni, Alessandra; Marcia, Loredana; Puddu, Alessandro; Cao, Antonio; Schlessinger, David; Crisponi, Laura
The FOXL2 forkhead transcription factor is expressed in ovarian granulosa cells, and mutated FOXL2 causes the blepharophimosis, ptosis and epicanthus inversus syndrome (BPES) and predisposes to premature ovarian failure. Inactivation of Foxl2 in mice demonstrated its indispensability for female gonadal sex determination and ovary development and revealed its antagonism of Sox9, the effector of male testis development. To help to define the regulatory activities of FOXL2, we looked for interacting proteins. Based on yeast two-hybrid screening, we found that FOXL2 interacts with PIAS1 and UBC9, both parts of the sumoylation machinery. We showed that human FOXL2 is sumoylated in transfected cell lines, and that endogenous mouse Foxl2 is comparably sumoylated. This modification changes its cellular localization, stability and transcriptional activity. It is intriguing that similar sumoylation and regulatory consequences have also been reported for SOX9, the male counterpart of FOXL2 in somatic gonadal tissues. PMID:20209145
Foxp1-mediated programming of limb-innervating motor neurons from mouse and human embryonic stem cells
Adams, Katrina L.; Rousso, David L.; Umbach, Joy A.; Novitch, Bennett G.
Spinal motor neurons (MNs) control diverse motor tasks including respiration, posture and locomotion that are disrupted by neurodegenerative diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy. Methods directing MN differentiation from stem cells have been developed to enable disease modelling in vitro. However, most protocols produce only a limited subset of endogenous MN subtypes. Here we demonstrate that limb-innervating lateral motor column (LMC) MNs can be efficiently generated from mouse and human embryonic stem cells through manipulation of the transcription factor Foxp1. Foxp1-programmed MNs exhibit features of medial and lateral LMC MNs including expression of specific motor pool markers and axon guidance receptors. Importantly, they preferentially project axons towards limb muscle explants in vitro and distal limb muscles in vivo upon transplantation�hallmarks of bona fide LMC MNs. These results present an effective approach for generating specific MN populations from stem cells for studying MN development and disease. PMID:25868900
A Method to Identify and Isolate Pluripotent Human Stem Cells and Mouse Epiblast Stem Cells Using Lipid Body-Associated Retinyl Ester Fluorescence
Muthusamy, Thangaselvam; Mukherjee, Odity; Menon, Radhika; Megha, P.B.; Panicker, Mitradas�M.
Summary We describe the use of a characteristic blue fluorescence to identify and isolate pluripotent human embryonic stem cells and human-induced pluripotent stem cells. The blue fluorescence emission (450�500�nm) is readily observed by fluorescence microscopy and correlates with the expression of pluripotency markers (OCT4, SOX2, and NANOG). It allows easy identification and isolation of undifferentiated human pluripotent stem cells, high-throughput fluorescence sorting and subsequent propagation. The fluorescence appears early during somatic reprogramming. We show that the blue fluorescence arises from the sequestration of retinyl esters in cytoplasmic lipid bodies. The retinoid-sequestering lipid bodies are specific to human and mouse pluripotent stem cells of the primed or epiblast-like state and absent in naive mouse embryonic stem cells. Retinol, present in widely used stem cell culture media, is sequestered as retinyl ester specifically by primed pluripotent cells and also can induce the formation of these lipid bodies. PMID:25068130
Neuropeptide Y receptor genes on human chromosome 4q31-q32 map to conserved linkage groups on mouse chromosomes 3 and 8
Lutz, C.M.; Frankel, W.N. [Jackson Lab., Bar Harbor, ME (United States)] [Jackson Lab., Bar Harbor, ME (United States); Richards, J.E. [Univ. of Michigan Medical School, Ann Arbor, MI (United States)] [and others] [Univ. of Michigan Medical School, Ann Arbor, MI (United States); and others
Npy1r and Npy2r, the genes encoding mouse type 1 and type 2 neuropeptide Y receptors, have been mapped by interspecific backcross analysis. Previous studies have localized the human genes encoding these receptors to chromosome 4q31-q32. We have now assigned Npy1r and Npy2r to conserved linkage groups on m