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Immunoglobulin knockout chickens via efficient homologous recombination in primordial germ cells
Edited by Max D. Cooper, Emory University, Atlanta, GA, and approved October 30, 2013 (received for review September 12, 2013)
Chickens have been an important animal model in the fields of developmental biology and immunology over the last century and have contributed a number of basic findings in these areas. However, their use has been limited more recently because there has been no way to genetically engineer specific mutations in the chicken. In addition to basic research applications, genetic modification in birds could also be used to mitigate the threat of avian influenza by production of flu-resistant bird stocks in agriculture. Here we describe an efficient way to target genes of interest in the chicken genome and create knockout chickens. We targeted the immunoglobulin heavy chain gene, leading to loss of antibody production and a block in B-cell development.
Gene targeting by homologous recombination or by sequence-specific nucleases allows the precise modification of genomes and genes to elucidate their functions. Although gene targeting has been used extensively to modify the genomes of mammals, fish, and amphibians, a targeting technology has not been available for the avian genome. Many of the principles of humoral immunity were discovered in chickens, yet the lack of gene targeting technologies in birds has limited biomedical research using this species. Here we describe targeting the joining (J) gene segment of the chicken Ig heavy chain gene by homologous recombination in primordial germ cells to establish fully transgenic chickens carrying the knockout. In homozygous knockouts, Ig heavy chain production is eliminated, and no antibody response is elicited on immunization. Migration of B-lineage precursors into the bursa of Fabricius is unaffected, whereas development into mature B cells and migration from the bursa are blocked in the mutants. Other cell types in the immune system appear normal. Chickens lacking the peripheral B-cell population will provide a unique experimental model to study avian immune responses to infectious disease. More generally, gene targeting in avian primordial germ cells will foster advances in diverse fields of biomedical research such as virology, stem cells, and developmental biology, and provide unique approaches in biotechnology, particularly in the field of antibody discovery.
The chicken has historically been an important model vertebrate organism in the fields of developmental biology and immunology and has contributed a number of basic tenets to these fields. For example, B lymphocytes were first recognized in chickens as the antibody-producing cells and are named after the bursa of Fabricius, a gut-associated lymphoid tissue (GALT) that is required for B-cell development in chickens (1). Graft-versus-host response was first described in chicken embryos (2), and the first attenuated vaccine was developed by Louis Pasteur against fowl cholera caused by Pasteurella multocida. Nevertheless, the lack of a robust genome editing technology including knockouts has put the chicken at a distinct disadvantage to mammalian species, especially the mouse, as a vertebrate animal model. The discovery of ES cells provided a powerful method to make desired changes to genes of interest in the mouse using homologous recombination, but interestingly, ES cells have not been as easily derived from other species. In the case of chickens, ES cells can contribute to all somatic lineages in high-grade chimeras, but germ-line transmission has not yet been demonstrated, precluding their use in creating fully transgenic chickens (3). Although ES cells are not germ line competent in chickens, embryo-derived primordial germ cells (PGCs) can be cultured indefinitely, transfected, clonally selected, and reintroduced into the embryo where they colonize the gonad and give rise to fully transgenic progeny in the next generation (4⇓⇓–7). Thus, in chickens, cultured stem cell lines are specific for somatic lineages or the germ line but not both as with mouse ES cells.
Transgenic chicken technology has a number of important applications in the pharmaceutical and agricultural industries. The creation of influenza-resistant chickens could result in a huge benefit to human health. Transgenic chickens overexpressing a short hairpin RNA that acts as a decoy for the influenza polymerase were shown to block the transmission of virus, leading to a strategy for controlling avian influenza outbreaks (8). Transgenic hens have also been made for protein production in the egg using egg white–specific promoters to drive expression in the oviduct (9, 10). Large-scale production of therapeutic proteins that are of interest for the pharmaceutical industry can be produced in a time- and cost-efficient manner. Another human health-related application is creation of transgenic chickens expressing human antibodies, similar to "humanized" mice, rats, and cattle (11⇓⇓–14). Such chickens would take advantage of the phylogenetic distance between chickens and humans for the discovery of human therapeutic antibodies against targets that are highly conserved between mice and humans but immunogenic in avian species.
The first transgenic chicken was produced by direct DNA injection into the fertilized egg (15), a very inefficient process in which the transgene insertion site cannot be controlled. The efficiency increased with the use of retroviral (13, 16, 17) and lentiviral vectors (18), and more recently with transposons (4, 5), but targeting and more subtle mutations are not possible with these approaches. Here we show that classical gene targeting by homologous recombination can be efficiently executed in cultured chicken primordial germ cells to generate knockout birds. In this study we chose to target the Ig heavy chain J gene segment (JH) to create a B cell–less chicken.
Many attempts have been made in the past to generate a B cell–less chicken. Surgical removal of the bursa at an early stage results in depletion of the B-cell lineage and a permanent reduction in antibody production, although results can vary because surgical bursectomy is technically challenging and it is difficult to ensure complete removal of B cells and secreted Ig (19). In chickens, unlike mammals, there is a developmental window during which the Ig genes rearrange, and the adult B-cell population is entirely derived from cells that undergo rearrangement during embryonic and early postembryonic life (20). Also at this time, B-lineage cells colonize the bursa, and there is a rapid expansion of cells expressing surface Ig (sIg). Experiments with chimeric B-cell receptor (BCR) proteins have indicated that basal BCR signaling is required for early stages of B-cell development in the bursa (21⇓–23), although the lack of knockout technologies in chickens has precluded the analysis of deletion of specific BCR components in vivo.
Generation of a B cell–less chicken by knockout of the Ig JH segment now overcomes this problem. The opportunity to study aspects of immunity better in JH knockout chickens will help to improve vaccine technology for the poultry industry. More generally, the availability of knockout chickens demonstrates that targeting technologies long established in mice are now available in birds.
Deletion of the Ig JH Segment.
To create a null allele of the chicken IgH locus we deleted the single known JH segment and its recombination signal sequences by homologous recombination in chicken PGCs. Although the first draft chicken genome was published in 2004, the IgH sequences remain incompletely characterized and are found only in small, unordered contigs (24⇓–26). Regions flanking the JH segment were cloned by PCR using primers spanning the gaps in the genome assembly, from the cell line we used for targeting (derived from a cross of commercial Brown Leghorn and the Minnesota Marker Line), and homology regions totaling ∼8 kb were assembled into an isogenic DNA targeting vector with a puromycin resistance cassette and Enhanced Green Fluorescent Protein (EGFP) flanked by loxP sites (Fig. 1A). To facilitate future insertion of foreign genes such as human Ig sequences into the targeted IgH locus, an attP site and promoterless neo gene were included for site-specific recombination by the phiC31 integrase. The apparent targeting frequency in PGCs with this vector was high; 7 of 25 puromycin-resistant clones screened had a correctly targeted event (28%). This high frequency may reflect the fact that randomly integrated (nontargeted) clones are suppressed, possibly from silencing of the drug selectable marker when inserted in most genomic sites in these germ-line cells (27). The absolute frequency was about one targeted clone per 107 transfected cells, which is in the range of mouse ES cells (10−5–10−8). Germ-line transmission of JH knockout (JH-KO) clones was lower than previously reported with random insertion transgenes (Fig. 1D). Once established in fully transgenic animals, the knockout allele was inherited in Mendelian fashion, and Southern blotting confirmed a single, correct integration of the vector in the IgH locus (Fig. 1C). To easily genotype offspring from heterozygous matings, a PCR assay was established using a primer pair in the deleted J region and a second primer pair spanning from the selectable marker cassette into the 3′ homology region (Fig. 1B).
Depletion of Peripheral B Cells and Plasma Ig in JH Knockout Birds.
On day 7 after hatch, the homozygous JH-KO birds showed a highly significant reduction in IgM levels in comparison with the control groups (Fig. S1). IgY levels were high for all three genotypes because maternal IgY transmitted via the egg yolk persists for several weeks. Five weeks after hatch, both the IgM and IgY levels of the JH-KO/JH-KO birds were indistinguishable from the PBS control (Fig. 2A). In the heterozygotes, IgM was reduced at day 7 compared with WT chickens (Fig. S1), and by day 35, there was a statistically significant reduction in the IgY levels, although IgM levels were normal (Fig. 2A). Because the process of allelic exclusion guarantees that each B cell expresses only one allele in WT B cells, these data were contrary to our expectation.
We evaluated the cellular composition of the peripheral blood lymphocytes, the bursa, and the spleen 3 wk after hatch by flow cytometry. In the spleen and blood, the B-cell population was essentially deleted in the JH-KO/JH-KO birds [less than 0.2% Bu-1 (a pan-B-cell marker), 0.08% IgM, and 0.2% IgL positive cells were detectable], whereas normal levels were observed in WT and JH-KO/+ chickens (Fig. 2B). No significant difference was measurable in the T-cell or thrombocyte populations (Fig. 2B). At three weeks after hatch, the bursas of JH-KO/JH-KO birds contained few living lymphocytes; as a consequence, we analyzed the bursa 3 d after hatch. Bu-1– and CD45-positive cells were reduced to 67.7% and 84%, respectively, in the homozygotes, compared with ∼98% in the control groups. The reduced proportion of Bu-1+ and CD45+ cells in the bursa could reflect the fact that the cells are undergoing apoptosis and possibly down-regulating expression of these markers. No surface IgM was detected in the homozygotes (Fig. 2B). The B-cell lineage of bursal sIg-negative cells in the homozygotes was confirmed by finding that the light chain locus is rearranged and undergoing gene conversion in these cells, without selection for productive rearrangement (Figs. S2 and S3).
Silencing of eGFP in the Heavy Chain Locus.
Not all somatic cells of the transgenic birds were EGFP positive by FACS, despite flanking copies of the β-globin insulator and expression driven by the β-actin promoter. The proportion of EGFP-positive cells ranged from 30% to 50% in heterozygous and homozygous birds. As there was no evidence for loss of the EGFP gene by Southern blot analysis, we looked for gene specific silencing of the EGFP in chicken embryo fibroblasts (CEFs) from JH-KO/+ birds. After limiting dilution, we compared the CpG methylation status of the EGFP gene in a cell pool with 2.5% EGFP-positive cells vs. a pool with 60% EGFP-positive cells by bisulfite sequencing. We found that the CpG methylation status of specific sites in the EGFP gene correlated with EGFP expression. At one CpG site analyzed, 84% of all sequences showed methylation in the low EGFP CEF pool (Fig. S4), whereas only 7% were methylated in the high EGFP CEF pool (Fig. S5). EGFP silencing has not been reported in other lines of transgenic birds and may be a consequence of its position in the heavy chain locus.
Histological Analysis Shows Depletion of Lymphocytes in the Bursa of JH Knockout Birds.
Progeny of matings between JH-KO heterozygotes hatched normally, and homozygous JH-KO/JH-KO birds grew at the same rate as their hatchmates (Fig. 3A). The weights of the bursa and the spleen were drastically lower in the JH-KO/JH-KO compared with JH-KO/+ and WT birds by 28 d posthatch, although at embryonic day 18, there was no difference in the weights of the spleens and bursas in the mutant genotype (Fig. 3 B–D). Histological sections of bursas were prepared at several stages to evaluate morphology. At embryonic day (ED)14, ED18, and day 1 after hatch, colonization of the epithelial buds was seen in all genotypes, and no defect was apparent in the homozygotes. By 4 wk after hatch, WT and JH-KO/+ knockout birds showed a clear separation of follicle medulla and cortex by cells migrating across the basement membrane that forms the cortico-medullary junction, whereas no such separation was apparent in the JH-KO/JH-KO birds (Fig. 4). This finding was supported by staining the cortico-medullary junction with a cross-reactive anti-human desmin antibody that specifically stains the basement membrane (Fig. 5). Bursal follicles of the JH-KO/JH-KO birds were reduced in size, and although immunohistochemistry with Bu-1 shows that B-lineage cells were still found in the follicles of JH-KO/JH-KO birds at this stage, fewer cells were present compared with follicles from age-matched control birds (Fig. 6A). However, IgM staining was completely absent in bursa and spleen sections of JH-KO/JH-KO birds (Fig. 6). Some Bu-1–positive cells were detectable in spleen sections from JH-KO/JH-KO birds (Fig. 5B); this could be due to the possible cross-reactivity of the antibody to macrophages (28). The CD3-positive T-cell zones in the spleens of JH-KO/JH-KO chickens were well defined even in the absence of B cells (Fig. 6B).
Lack of Antibody Response on Immunization of JH Knockout Chickens.
To determine if JH segment knockout birds can produce antibodies after immunization, hens were immunized at 5 wk of age with keyhole limpet hemocyanin (KLH). WT and JH-KO/+ knockout birds showed increasing IgM titers by ELISA against KLH 3 and 5 d after immunization (Fig. S6). KLH-specific IgY was detectable in WT and JH-KO/+ 5 d after immunization (Fig. S6). No reactivity toward KLH was seen in the IgM or IgY ELISA in plasma from JH-KO/JH-KO chickens (Fig. S6).
The ability to culture PGCs that retain germ-line competence once they are injected back into host embryos is unique to chickens. By combining cultured PGCs and classical gene targeting via homologous recombination, it is now possible to introduce a site-specific alteration into the chicken genome. The targeting frequency is high when measured as the ratio of targeted clones to the total number of drug resistant clones, making screening for knockout clones simple and efficient. The absolute targeting frequency requires transfection of at least 107 cells to get one targeted clone. This number of cells is readily obtainable within 40 d following isolation of about 200 PGCs from a single embryo. Because cultures originate from a single embryo, they can be used for targeted modification with isogenic DNA. PGC lines may be grown for more than 150 d in culture without loss of germ-line competence (6), thereby providing a nearly limitless resource of cells. In an initial experiment a shorter 3′ homology region of 3 kb was used, which led to a somewhat lower targeting frequency of 10% (3 targeted clones out of 28 screened). The lower limit of the length of the homology arms has not been established.
EGFP was included in the selectable marker cassette of the knockout vector to facilitate identification of genetically modified cells in culture and to select chimeras for breeding to obtain G1 transgenic offspring by measuring the proportion of GFP-positive sperm in semen via flow cytometry. We also placed an attP site joined to a promotorless neomycin resistance gene into the chicken heavy chain locus (Fig. 1A) to facilitate integrase-mediated targeted insertion into the heavy chain locus to express, for example, V regions derived from the human Ig heavy chain locus. Replacement of both the heavy and light chain V regions would yield chickens from which fully human antigen-specific antibodies could be isolated. A humanized chicken will provide a major advance over mammalian-based antibody discovery platforms. The chicken has not shared a common ancestor with humans in more than 300 million years. As a consequence, many human antigens that are not immunogenic in mammals because of their high homology will give a strong antibody response in chickens. Empirical data from our laboratory have demonstrated that unique epitopes can be accessed from immunized WT chickens. In combination with the ability to make site-specific insertions via the attP site targeted into the IgH locus, it will be possible to evaluate the epitope coverage of chickens making fully human antibodies.
The use of homologous recombination in primordial germ cells has several advantages over nascent technologies such as sequence-specific nucleases for making targeted changes to the genome. Although the in vivo use of zinc finger nucleases and transcription activator-like effector nucleases (TALENs) has been suggested by Tyack et al. (29) their use requires that the genetically modified genotype be identified in hatched chickens and not in cultured cells. From a practical perspective, it is currently more attractive to screen for the genotype in culture than in live chicks, although newer technologies such as clustered regularly interspaced short palindromic repeats (CRISPRs)-Cas9 may change this calculation. The choice of technologies is equally influenced by the flexibility to insert selection cassettes, genetic markers such as GFP and recombination target sequences such as attP, which is best accomplished in cultured cells. There is no advantage in chicken PGCs to use a sequence-specific nuclease when inserting selection cassettes because the targeting frequency is already high. The avian-specific follicular anatomy makes it impractical to directly inject chicken zygotes with genome editing tools, and in vivo transfection of embryos is likely to be very inefficient.
Deletion of the JH gene segment in chickens results in a total loss of heavy chain expression, proving that the chicken genome harbors a single functional heavy chain locus. The strategy of targeting the single JH segment ensures that all heavy chain expression is blocked, because it is required for all heavy chains regardless of V region use or isotype class. In the knockouts, the B-cell receptor complex is not required for population of bursal follicles, in seeming contrast to the previous conclusion that signaling by the B-cell receptor component Igα is required (30). B cells in the JH knockout chickens not only colonize the bursa, but they also proliferate inside the follicles. The epithelial buds are colonized with approximately three B cells per follicle, and these cells proliferate until the bursa contains ∼107 B cells (20). Because bursal morphology and bursal weights at the time of hatch are indistinguishable between KO and WT birds, it appears that B-cell receptor signaling is not necessary for proliferation inside the B-cell follicles. Light chain rearrangement and gene conversion proceed in the bursa of the knockouts, indicating that these diversification processes are also independent of IgH expression. Only one-third of the rearranged light chain genes are in-frame, which is consistent with the absence of selection for productive rearrangement required by a functional receptor. After hatch, expression of the heavy chain becomes obligatory for continued B-cell survival, and by 4 wk of age, bursal follicles in the JH-KO/JH-KO chickens are depleted of B cells. More detailed studies of the molecules required for BCR signaling in B-cell development can now be designed using the IgH knockout birds in experiments that will extend previous work done in WT birds (30⇓⇓–33).
The availability of chickens without B cells will also find utility in both avian and human medicine. In chickens, several diseases including Marek's disease, infectious bursal disease, avian leukosis, and chicken anemia virus express an immune-compromised phenotype. The IgH knockout chickens described in this paper are currently being developed as a research tool to better understand the etiology of these diseases. In human medicine, avian influenza is a significant zoonotic disease infecting both humans and chickens. The IgH knockout will be incorporated into the toolbox of virologists developing technologies to obviate the anticipated pandemic of increasingly virulent recombinant viruses.
The chicken has a long and distinguished career in fields as diverse as immunology, developmental biology, genetics, animal breeding, agriculture, nutrition, virology, oncology, and biochemistry. In addition, chickens supply approximately one-third of the animal protein in human diets throughout the world. Here we show site-specific changes to the chicken genome, which in combination with the chicken genome sequence and several approaches to nontargeted genetic modification, completes the range of genetic modifications that can be applied to avian models in biomedical research and to poultry production.
Animal Experiments.
Commercial Brown and White Leghorn chickens and Minnesota Marker Line chickens were used. Birds were housed on short days (8 h) in groups of four birds per cage up to an age of 12 wk. Males and females were moved at 12 and 17 wk of age, respectively, to long days (14 h) into individual cages. All birds received standard diet and water ad libitum. Animal experiments were done in accordance to Institutional Animal Care and Use Committee (IACUC) approved protocols and under supervision of the IACUC.
Construction of Targeting Vector.
To target the chicken JH segment, ∼9 kb of genomic sequence surrounding JH was cloned and sequenced. Genomic DNA was obtained from the cell line used for the gene targeting transfections (Nu69) and used in PCR to amplify heavy chain sequences upstream (2.3 kb) and downstream (6.3 kb) of JH. Extensive polymorphism between the two alleles in Nu69 was observed (one allele is from Brown Leghorn and one is from the Minnesota Marker Line), and care was taken to assemble a single contig from one of the two alleles for targeting. Targeting vector IgH KO2 was assembled with a 5′ homology region of 1938 bp (delineated by primers 5′ HR-for, 5′-TGGGAAATTTGGCCCTCTTGGCC-3′ and 5′ HR-rev, 5′-TCGGGGCGAAAACCGCCCATTT-3), a 3′ homology region of 6,070 bp (delineated by primers 3′ HR-for, 5′- TGGCGGTCTGAGGGGAAAATGTC-3′ and 3′ HR-rev, 5′-ATATTGGCCCCATTTCCCCTCAG-3′), and a selectable marker cassette consisting of β-actin-EGFP and CAGGS-puromycin flanked by duplicated copies of the core 300-bp HS4 insulator from the chicken β-globin gene. LoxP sites flank the HS4-EGFP-puro cassette. Adjacent to the main cassette is a promoterless neomycin resistance gene linked to an attP site for targeted insertion of attB-containing plasmids using phiC31 integrase.
PGC Derivation and Culture.
The parental line Nu69 was derived from a cross of the Minnesota marker line with a commercial Brown Leghorn line. Primordial germ cells were derived from blood collected at stages 13–15 (Hamburger-Hamilton) from a male embryo and cultured in KO-DMEM containing 40% (vol/vol) Buffalo rat liver (BRL) cell conditioned medium, 7.5% (vol/vol) FBS, 2.5% (vol/vol) chicken serum, 2 mM glutamine, 1 mM pyruvate, 1× nonessential amino acids, and 0.1 mM β-mercapto-ethanol and supplemented with 6 ng/mL recombinant mouse stem cell factor and 4 ng/mL recombinant human fibroblast growth factor basic (R&D Systems) on a feeder layer of irradiated BRL cells (6, 7).
Transfection of PGCs.
Targeting construct IgH KO2 was linearized using a unique NotI restriction site at the 3′ end of the 3′ homology region before transfection; 5 × 106 cells were suspended in 100 µl of AmaxaV buffer with 20 µg linearized DNA and transfected as previously described (6, 7). Each transfection was plated on a full 48w plate. Puromycin selection (0.5 µg/mL) was started 3–5 d after transfection, and medium was changed regularly throughout selection. Puromycin-resistant clones were expanded for genotyping and injection.
Injection of PGCs.
Recipient embryos were incubated until stages 13–15 (Hamburger-Hamilton) and transferred to a collection dish. After injection of 3,000 JH-KO PGCs into the vasculature (6, 7), the embryos were transferred to a surrogate shell and incubated until hatch.
Southern Blot.
The 5′ external probe was amplified and confirmed by sequencing from Nu69 primordial germ cell gDNA using the following primers: 5′ CAGTGTCCAAATTCCTTAAATTTCC 3′ and 5′ TCGTTGTGAAATGCGGTGAAAAT 3′. The EGFP probe was prepared from a plasmid containing EGFP by SalI/NotI digest followed by gel purification. For Southern blot analyses, 5 µg gDNA was digested for 12 h with EcoRI. Digested gDNA was separated by gel electrophoresis and visualized by poststaining with GelRed (Biotium). Depurination was done by incubation with 0.2 M HCl for 10 min, followed by denaturation in 0.5 M NaOH and 1.5 M NaCl. The gel was neutralized by two washes in 1 M Tris, pH 7.4, and 1.5 M NaCl. Transfer to Amersham Hybond-N+ blotting membrane (GE Healthcare) was done overnight by capillary transfer. After transfer, the gDNA was cross-linked using a UV light. Membranes were prehybridized at 65 °C for 1 h with Rapid-hyb buffer (GE Healthcare). Probes were labeled using the Prime-a-Gene Labeling System (Promega) following the manufacturer's protocol. After hybridization for 2 h, blots were washed once with 2× SSC and 0.1% SDS, followed by two washes with 0.1× SSC and 0.1% SDS. Blots were exposed to film for 24–96 h at −80 °C.
For genotyping, 5 µL blood was used for gDNA isolation using the DNeasy Blood & Tissue Kit (Qiagen). PCR primers specific for the JH-KO were as follows: neo-R1, 5′AGTGACAACGTCGAGCACAGCT 3′; JC-R45, 5′GCCCAAAATGGCCCCAAAAC 3′. PCR primers specific for the deleted WT region were forward primer JH-F3 5′ATGGGGCCACGGGACCGAA 3′ and JC-R45 (above). PCR was performed using 5× FirePol MasterMix (Solis BioDyne) according to the manufacturer's instructions.
Hens were immunized by intramuscular injection at 5 wk of age with 300 µg KLH (Sigma Aldrich) mixed 1:1 with Freund's complete adjuvant (Sigma Aldrich). One boost was performed 2 wk after initial immunization using 300 µg KLH mixed 1:1 with Freund's incomplete adjuvant (Sigma Aldrich).
To measure total plasma IgM and IgY, enzyme immunoassay (EIA) plates (Corning) were coated with 2 µg/mL goat anti–chicken-IgM (Bethyl Laboratories) or rabbit anti–chicken-IgY (Sigma Aldrich) overnight. Plates were blocked with 3% (wt/vol) skim milk and incubated with chicken plasma for 2 h. Bound chicken IgM or IgY was detected with goat anti–chicken-IgM-HRP (Bethyl Laboratories) or rabbit anti–chicken-IgY-HRP (Sigma Aldrich) and developed with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate. OD was measured at 450 nm wavelength. All samples were measured in duplicate. To measure the IgM or IgY response after immunization with KLH, plates were coated with 10 µg/mL KLH. All subsequent steps were performed as for detection of total plasma IgM or IgY.
Cells (1 × 106) were stained for 20 min on ice using the following antibodies: mouse anti–chicken-Bu-1 (AV20), mouse anti-chicken-Cμ (M-1), mouse anti–chicken-lambda (L-1), mouse anti–chicken-TCRγδ (TCR1), anti–chicken-TCRαβ/Vβ1 (TCR2), mouse anti–chicken-TCRαβ/Vβ2 (TCR3), and mouse anti–human-CD51/61 (23C6) (Southern Biotech). Following a wash, all primary antibodies were detected by incubation with a goat anti–mouse-IgG-Cy5 (Bethyl Laboratories) antibody for 20 min. After a final wash, fluorescence was measured using a Beckman-Coulter FC500.
Tissue samples were either fixed with 10% (vol/vol) neutral buffered formalin for 24 h or immediately frozen in liquid nitrogen. Formalin-fixed samples were dehydrated and paraffin embedded, and 5-µm sections were collected and stained with H&E (EMS). For antibody staining, 10-µm sections were prepared from fresh frozen samples. Sections were rehydrated and fixed with ice-cold acetone. For the anti-desmin staining, endogenous peroxidase was blocked by incubation with 40% (vol/vol) methanol containing 0.3% H2O2 for 30 min. Nonspecific binding sites were blocked using the Vector ABC Kit and Biotin/Avidin Blocking Kit (Vector) following the manufacturer's instructions. Mouse anti-desmin (D33) (Thermo Scientific) was used as a primary antibody and detected using the Vector ABC Kit followed by the Vector DAB Kit (Vector). Eukitt mounting medium (EMS) was used to mount the stained sections. For all other staining, sections were blocked with 1% PBS-BSA. The following primary antibodies were used: mouse anti–chicken-Bu-1 (AV20), mouse anti–chicken-Cµ (M-1), and mouse anti–chicken-CD3 (CT-3) (Southern Biotech). After washing, the primary antibodies were detected using a goat anti–mouse-Cy3 (Bethyl Laboratories) antibody. Prolong mounting medium containing DAPI was used to mount the stained sections (Life Technologies). Pictures were taken using a Zeiss AxioImager M1 equipped with a Hamamatsu Orca 03 and QImaging MicroPublisher.
Methylation Analysis.
To analyze methylation in the EGFP gene, CEFs were prepared from 10-d-old heterozygous JH segment knockout embryos by trypsin digest. CEFs were cultured using Iscove's modified DMEM supplemented with 10% (vol/vol) FBS, 5% (vol/vol) chicken serum, and 1% penicillin/streptomycin. Cells were passaged every 2 d. Limiting dilution was performed, and predominantly EGFP-positive and -negative cultures were grown up. gDNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer's manual. Afterward, gDNA was treated with bisulfite using the EpiTect Fast Bisulfite kit (Qiagen). PCR was performed using the following methylation-specific primers: forward 5′ TTGTTCGTGTTTTGGTTTATTTTC 3′ and reverse 5′GCGAATCTTATAATTACCGTCGT 3′. The PCR product was cloned using a TOPO TA cloning Kit (Life Technologies). Bacterial colonies were sent for sequencing. Sequences were analyzed using DNASTAR Lasergene.
We thank Connie Cepko (Harvard Medical School, Boston, MA) for the CAG-EGFP construct. This work was supported by National Institutes of Health Small Business Innovation Research Grant GM090626 (to P.A.L.) and Research Fellowship Schu2446/2-1 (to B.S.) from the Deutsche Forschungsgemeinschaft.
- ↵1To whom correspondence should be addressed. E-mail: pal{at}crystalbioscience.com.
Author contributions: B.S., E.J.C., K.C.K., B.K., W.D.H., R.J.E., and P.A.L. designed research; B.S., E.J.C., H.Y., S.M.I., J.F., D.P., M.-C.v.d.L., and P.A.L. performed research; B.S., E.J.C., M.-C.v.d.L., and P.A.L. analyzed data; and B.S. and P.A.L. wrote the paper.
Conflict of interest statement: The authors have patents in process regarding the heavy chain knockout in chickens.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1317106110/-/DCSupplemental.
Freely available online through the PNAS open access option.
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This will be my 19th year teaching at Groveland. Most of my expereinces are in grades 5 and 6. I also teach a social studies methods class at the University of St. Thomas. I am passionate about reading, teaching, traveling, and being outside. When I am not at school I spend time with my family, dog, play soccer, and snowboard. If you have any questions please feel free to contact me, I prefere email.

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Media StudioOur one-of-a-kind studio has a news desk with lighting and video camera for our student newscast, a music studio for students to write and record their own music, and specialized therapy equipment to work with students with low vision and with students on their switch access skills.


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Saturday, 10-15-2016
Saturday Schedule
8:00am Class for Alpine members only please
9:00am & 10:00am Classes – Alpine and free class to family, friends and public. Come try us out complimentary with no obligations – today is a great first timer workout!
Workout of the Day
6 Rounds
1 Minute Air Assault Bike Calories
1 Minute Dumbbell Hang Clean to Push Press 35/25
1 Minute Barbell Thrusters 75/55
1 Minute REST
*Score total number of reps
*if more than 4 people in class, start at a different station including rest station


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If Novelty Revisions offered a virtual writer's mentoring program, where more experienced writers would be paired with newer writers to help them build confidence and push them to write, would you join?
I ask because the idea crossed my mind earlier this week. I'm not making any promises, and I would need to gauge interest before actually putting something like that together. But the subject of mentoring is one I have only covered from one side: that of being mentored. What about being a mentor?
Sometimes, as much as I love giving out writing advice in this format, not everyone wants it. Those who visit and comment often do – and I appreciate that, believe me. I suppose it's the byproduct of having had an amazing creative writing mentor when I was just starting out. I want to be able to be there for someone, writing wise, the way he was there for me.
But why? Why, when we have our own things to write and our own lives to live, should we even consider taking time and energy out of our busy schedules to act as a mentor to another writer?
Because the world needs more fearless writers.
Because too many people are being told they cannot create what they want to create. Too many people are stuck trying to do this on their own with no idea where to start. Most writers are busy – and it's true that you cannot improve as a writer if you do not go off on your own and write. But what about the aspiring writers who just don't have that kind of discipline? How can we help them learn? We can, can't we?
We should never be allowed to forget where we came from.
From a mentoring perspective, spending time with someone who looks up to you is an often much-needed reminder that you may not be perfect, but you have come much father than you once thought you could. There was a point in your life when you had never written down a story before. At this point in your life you might not remember what that was like. But having someone there who is in a place you once were, you are able to look back on that time in your life and better appreciate how hard you worked to get to where you are today.
We know what we're doing. Mostly. We can't let that go to waste.
Many of us have been writing for a long, long time. I'm not that old, but even I have pretty much been writing for over a decade now. I may not be an expert, but I know well enough the ins and outs of the industry to be able to answer many of the questions many new writers have when they are first starting out. Even if I don't have the answers, I have a decade's worth of resources to refer them to for help. Newer writers don't have any of the experience many of us have. Even if we are not "experts," we still have valuable advice to offer. Even simple things can mean the world to someone who is unsure and doubtful about what they can and cannot do.
Have you ever had an opportunity to mentor a less experienced writer? Would you want one? If enough of you like the idea, I'll see what I can do. Perhaps I will even recruit a few of you to help develop it. We shall see. :)
Meg is the creator of Novelty Revisions, dedicated to helping writers put their ideas into words. She is a freelance writer and an eight-time NaNoWriMo winner with work published in Teen Ink, Success Story, Lifehack and USA TODAY College. Follow Meg on Twitter.
Image courtesy of writingyorkshire.org.


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There is so much to think about and prepare for when you are pregnant, finding good stores that sell maternity clothes is not something you want to have to worry about. A lot of women also have trouble feeling comfortable or attractive while they are pregnant, so it is important to find the right stores that sell the clothing that can make you feel good all around. You want to find everyday clothing, plus some cute dresses and outfits that you can go out in that make you feel beautiful. Enjoy the list below.
Liz Lange Maternity at Target
Credit: Screenshot from Target.comTarget has a line of maternity clothing by Liz Lange. She started her collection when her pregnant friends couldn't find anything that they wanted to wear. Inspired to start designing, she built her company with just a few pieces, but then grew to dressing some of her favorite celebrities. She has learned how clothing should fit on pregnant women over the last ten years and her goal is to create cute clothing for pregnant women that they can enjoy wearing as much as she does designing it. In her collection there is something for just about any occasion including swimwear, dresses and skirts, sweaters and outerwear besides the typical tops and pants. Liz Lange Maternity at Target is a great suggestion.
Old Navy
Credit: Screenshot from oldnavy.gap.comAt Old Navy you can find a nice selection of quality maternity clothes that are affordable. Whether you need something for the office or you are just spending a casual day out, you can find something that is just right for their large collection of clothing for pregnant women. You can find workout clothes, attire for work, coats, shorts, and even swimsuits. At Old Navy they realize that your pregnancy is a special time in your life and that it shouldn't be complicated by dull or uncomfortable clothing. They strive to bring you fashionable clothing with their great designs and trendy looks so you can get all your favorites.
JC Penney
Credit: Screenshot from jcpenney.comIf you are not all that concerned about price, you can find another large selection of maternity clothing at JC Penney. A more sophisticated line, you will never be able to feel unattractive in maternity clothes from this store. They have all types of clothing for any occasion, plus they have coats and swimwear. There are tops and dresses that are as pretty as regular clothing, as they strive to bring their customers a look during pregnancy that they can feel good about wearing. If you are looking for support bands, they even have those, as well. When your baby is born they also have a selection of nursing bras as well.
Destination Maternity
Credit: Screenshot from destinationmaternity.comDestination Maternity is a shop that is focused on clothing especially for pregnant women. You can be assured that when you shop here you will find all types of clothing with you in mind. They offer collections including a pea in the pod, Jessica Simpson and Heidi Klum. You can easily shop their site by checking out the typical categories or choosing from things such as spring and winter styles, new items and lingerie. You can find clothes for work, active wear, outerwear and also sleepwear and sportswear.
Motherhood Maternity
Credit: Screenshot from motherhoodcanada.caMotherhood Maternity is where you will find a full selection of maternity clothing. From their stylish active wear to their selection of dresses that range from casual to classy, you will always be able to find what you are looking for here. Their dresses come in all different lengths depending on the occasion and all different sleeve styles as well from long sleeps to strapless. Keep yourself active during your pregnancy with their large selection of casual and workout wear, and find fun t-shirts with cute sayings such as "Bun in the Oven", "Princess in Progress" and "Baby Love".
To conclude this list of stores that sell maternity clothes, you shouldn't feel like you have to wear clothes during your pregnancy that are frumpy and out of style. You can find plenty of fashionable clothing that can make you feel good and look great as well. You won't even have to spend a lot of money on maternity clothing if you do not want to when you find the right stores that sell maternity clothes.


text: Santa Fe Title Company's staff is at your service and ready to help you list and sell all properties, close and settle all real estate transactions, and ultimately grow your business. We work by your side during every step of the process to ensure your success with each new home seeker and seller. In addition to full title underwriting and examination capabilities, full escrow and closing services, plus 1031 property exchanges, we provide the custom products and services you need to succeed. Consider us part of your real estate team that will help you meet all your business goals


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Why Namibia is failing to meet targets and implementing policies
Villager Reporters
When President Hage Geingob launched the Harambee Prosperity Plan in April 2016, he emphasised on a number of issues one of them being entrepreneurship and enterprise development. In the same spirit, Geingob changed the former trade and industry ministry to trade and industrialisation. He also appointed Penny Akwenye as his policy advisor on implementation and monitoring and promoted Tom Alweendo from National Planning Commission to director to minister of economic planning in the Presidency. When Calle Schlettwein succeeded Geingob as trade minister in 2012, he spoke about the need to establish industrial parks and presented to the National Assembly on 17 February 2015 the proposed implementation strategy for Namibia's Industrial Policy, the Growth at Home Strategy. Schlettwein said the Growth at Home was a theme chosen to reinforce the importance of accelerating economic growth, reducing income inequality and increasing employment. The government then adopted it as the country's first ever industrial policy meant to attain the strategic objectives for manufacturing as outlined in the 4th National Development Plan. These objectives, he said, would be supported by improved logistics and infrastructure, improvements in the ease of doing business and ongoing dialogue and partnership between government and the private sector. "Therefore, the debate on the industrial policy has shifted in recent years from the question of whether developing countries should or not seek to industrialise, to the question of how to best design strategies and subsequent intervention tools which can promote a process of inclusive and sustainable industrial development," Schlettwein told the National Assembly then.
Both President Hage Geingob and Schlettwein have spoken about establishing industrial parks, which are common in China. When he was the premier in 2013, Geingob hosted a Chinese delegation from the Non-Ferrous Metals Investment Holding who presented a proposal for iron ore mining and industrial park projects in the Kunene region. ECE had discovered a deposit of 3 268 billion tons of iron ore at the Orumana village, about 30km south of Opuwo. The company said the multi-billion dollar industrial park would be built near Opuwo and that when completed, the projects would contribute US$700 million (about N$6,2 billion) to the gross domestic product (GDP) of Namibia and employ about 5 000 Namibians.
According to the company, US$20 million in geological, geophysical prospecting, drilling and trench work had been invested at the time. The industrial park, the company said, would be based on iron ore mining and steel smelting, supported by related industries, including section bar processing, machinery, trade, logistics, hotels, among others. Schlettwein said the concept of an industrial park is a very exciting proposal. "We are looking at a large project to significantly create jobs, skills' development, wealth creation and value addition to our raw materials," he said at the time. In April 2014, Schlettwein said the trade ministry had set aside N$593 million in the 2014/15 budget for the construction of the Pioneer Industrial Estate at Walvis Bay. He also said a further N$893 million would be made available for the same project for 2015/16, while another N$611 million would be set aside for the development of an automotive and automotive spare parts. According to the documents, the industrial park would accommodate industries specializing in auto and spare parts; a high-value beef processing facility; beef export facility; value adding facilities for textile; wool; pelts; silk and wool; steel processing; manufacturing of metal products and the manufacturing of a variety of building materials and furniture manufacturing, pharmaceuticals and cosmetics.
The lack of space, according to the Namibia Manufacturers Association chief executive officer, Ronnie Varkevisser, has left their members fighting for shelf space with retailers especially those from the South African market. Varkevisser said the association has 153 members who contribute to both the primary and secondary sectors. They are into basic metals and fabricated metal products as well as other non-metallic mineral products such as rubber and plastic, coke refined petroleum products, pharmaceuticals and toiletries, pulp, paper products publishing and print, wood and wood products, leather and leather products, textile products as well as food and beverage products. Varkevisser said most Namibian producers often pay very high prices to have their products on foreign shelves whenever they export to warehouses in South Africa, where the goods then return to Namibia with a 'PROUDLY SOUTH AFRICAN' sticker.
Growth at Home Strategy
Over the years, Schlettwein has been at pains to push for implementation of the Growth at Home Strategy and value addition to enhance economic growth. Schlettwein, however, refused to answer questions sent by The Villager this week regarding the issues he pushed as the trade minister. The Villager wanted to know what
happened to the idea of industrial parks in Namibia that were supposed to have been built by the government and the Chinese. The other question was whether the Growth at Home Strategy was working. Schlettwein referred The Villager to the industrialisation, trade and small to medium enterprises minister Immanuel Ngatjizeko. "We wish to refer you to the Ministry of Industrialisation, Trade, and SME Development, who are the line custodian ministry as per your questions," Schlettwein said an emailed response. This is despite that he used to speak passionately about how Namibia with all its abundant natural resources, will not successfully grow its economy if the country does not start implementing its industrialisation policy in earnest. However, in his foreword to the 2015/16 annual report for the industrialisation ministry, Ngatjizeko said they have put in place the requisite legal and regulatory building blocks to fully industrialise Namibia. "Our strategic objectives are therefore to promote the country's manufacturing activity; to grow and diversify our exports and export markets; and to promote the growth and development of our SMEs," he said. The report titled Growth at Home says that the ministry had approved 15 companies and assisted four with various interventions ranging from technical assessments, coaching and
training, products development, market exposure and equipment purchases. According to the report, N$36, 2 million had been used to help 895 SMEs to acquire machinery and equipment under the Industrial Upgrading and Modernisation Programme to improve the production and supply capacity, efficiency and export competitiveness of local enterprises. Furthermore, the report says five SME parks were built under the sites and premises development programme at Okakarara, Ruacana, Keetmanshoop, Onethindi and Grootfontein to enable business operations. Because all these projects were completed during Schlettwein's time at trade, The Villager wanted Ngatjizeko to say what he has done at the ministry ever since he took over regarding progress on what was laid down for him. Ngatjizeko said industrialisation will depend on resources – financial and manpower-wise. "We think we are going to do our best in order to industrialise. Whether we will succeed by 2030 will depend to what extend we would be industrialised," he said. He did not, however, say anything about the Growth at Home Strategy. But Varkevisser said the loopholes in executing the Growth at Home Policy range from lack of incentives to local manufacturers and producers to lack of state support on foreign trade policies in countries that Namibia ex
ports to. He cited the challenges faced by Namibian salt producers in Brazil where there is a 25% marine tax, which despite efforts to have it removed by Namibia came to naught. "We are constantly advocating to government through the industrialisation ministry and Namibia Trade Forum as well as other stakeholders to push high level delegation discussions with the retailers who are not yet supporting any local manufacturer and thus execute the "Retail Charter" initiative so that they can start purchasing local products?" Varkevisser said.
Infant Industry Protection
The other thing Schlettwein defended was the adoption of the policy and regulatory measures regarding the protection of the infant industries. So far, between 2013 and 2014 Namibia has instituted infant industry protection (IIP) measures on agro-processing sector and specified poultry and dairy products. It is not clear whether these measures are still in place since the presidential advisor in charge of policy implementation, Penny Akwenye could not Speaking in the National Assembly on 14 February 2014, Schlettwein said the primary reason was to preserve and nurture the economic and industrial growth and in turn accelerate job and wealth creation and to equalise wealth distribution by cushioning and creating policy space for existing
economic value chains to get off the ground and build the requisite competitive capacity. "The global trading system poses a harsh environment for small developing countries. The cost of access to the global market has been to give up policy options through which to support, protect and nurture industries until they are sustainable and competitive," he said. He pointed out that there are massive subsidies paid by the developed economies in especially the agricultural and agro processing sectors, which continue to distort markets and make it very difficult for developing economies like Namibia's to develop and industrialise. Schlettwein further said that for Namibia to overcome low growth, poverty, high unemployment and income inequality, requires a paradigm shift and real restructuring and transformation of the economy. "In particular we need, on one hand, to transform the economy from its heavy reliance on the production and export of raw material commodities and, on the other hand, to become an industrialised economy where manufacturing, innovation and the development of value chains assume the centre stage. "We are able to add value to our resources and to produce some of the consumer goods that we import in large quantities. Vision 2030, the current national development plan (NDP4) and our national industrial policy provide a sound framework
for targeted action to bring about sustained and high levels of economic growth and employment, wealth creation and income distribution and equality," he said. Addressing the Growth at Home conference in Windhoek in 2013, Geingob, who was the premier then said: "In today's ever-changing economic and political global environment, the importance of an effective domestic law to safeguard our ownership and management of strategic natural resources cannot be overstated."
Doubts abound
DTA president McHenry Venaani said they have not yet started with the first phase of industrialisation. "First and foremost, we have to identify key-sectors in which we want to industrialise. By so doing you create the necessary human resource capacity to be able to venture into those sectors. "So you can't industrialise without human resources and we have not created that. I do not think we will make it in less than 13 years. There is no clarity. You start with value addition and processing. All countries that have moved towards industrialisation have achieved value addition. I do not think we are running the race at full speed. We still have a lot of work to do. It's just political rhetoric," he said. Opposition member Lukato Lukato said there is a lot of corruption going on from the top to the bottom of gov
ernment and that Vision 2030 will not be likely. "Government is spending a lot of money on foreign trips, taxpayers' money which should have been invested towards attaining the industrialisation vision. "What happened is that when they misused a lot of money and when they discovered that they have no money, they simply stopped the Industrial parks project," he said. University of Namibia economic analyst Roman Grynberg said the Chinese have put up industrial parks in Zambia, Ethiopia and in Mauritius, but with the exception of Ethiopia there have not been much success. "In Zambia again the project has not been that bad but it has not been a great success. It would be nice working with fellow African states to build industrial parks here but who? Who can you recommend? Rwanda, Burundi, Burkina Faso, Cameron plays good football, nobody has that money," he said.
The Villager Newspaper sought comment from Finance Minister Calle Schlettwein, National Planning Commission Minister Tom Alweendo who could not comment. The Villager also efforts to reach Tarah Shaanika whose phone was unreachable while Swapo SG Nangola Mbumba preferred not to comment.


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- Remove battery
- Unlock switch next to M 2.5x10
- Locate unlock switch in the grille, somwhere near center. Push it down and hold while pulling out the ultrabay.
Saturday, July 12, 2014
Removing Ultrabay Lenovo Y500
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The Batavia Kill watershed is located within three different townships: Middletown and Roxbury in Delaware County, and Halcott in Greene County. The drainage area of the Batavia Kill is approximately 19.3 square miles and the mainstem is 10.4 stream miles from the headwaters to the confluence with the East Branch Delaware River mainstem.
Roxbury Run, Denver and Vega are small population centers located in this sub-basin. There are two reaches in the basin where the valley is "pinched". The rest of the management units have valleys that are broad to very broad. The land use is predominately forested with some agricultural fields. The average annual rainfall in the watershed can range from 37-41 inches/year at the lower portion to 41-49 inches/year in the headwaters. The Dry Brook mainstem is a fourth order stream. There are numerous unnamed tributaries and two major tributaries that enter the mainstem: Bed Hollow and Buffalo Hollow.
CLICK HERE to access the Batavia Kill portion of the EAST BRANCH DELAWARE STREAM MANAGEMENT PLAN (scroll to page #65)
Current access includes NYCDEP lands on George Lawrence Road – hear the headwaters of the Batavia Kill.


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Dennis Cleveland Cobb
Dennis Cleveland Cobb, 64 years old, passed away on September 18th, 2011. He was born March 26th, 1947 in Miller, South Dakota.
Dennis was a loving husband, proud father and felt blessed to be called Papa. His greatest joy was spending time with his children and grandchildren. Dennis was a deacon at Texas Avenue Baptist , belonged to BAGMA and sang for a nursing home ministry for many years. Dennis was also a member of a Southern Gospel trio, the Christian Heirs. His love of life was for his Lord, family, singing and fishing. Dennis witnessed Christ' love through how he lived his life each day.
He is survived by his wife Donna of forty four years; daughter, Tammy Bruno and her husband Chad, son Chris, and daughter Becky; grandchildren, Hannah, Shane and Abby Bruno, Emily Ann, Amanda, Ian and Sarah Cobb; sister, Mary Jo Wallman.
Funeral Services will be held at 11:00 a.m., September 22nd, 2011 at Texas Ave. Baptist Church with Pastor Rudy Guerrero officiating. Burial will follow at 2:00 p.m., September 23rd, 2011 at the New Chatfield Cemetery in Chatfield, Texas.
Please feel free to post tributes at www.carnesfuneralhome.com


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Metro Business Alternatives
05/12/2016 | business opportunities | No Comments
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Hourly Consulting that Meets You Where You're At
Pintaya Consulting offers hourly consulting services to businesses looking for outside perspective on the performance of their accounts. Typically, this option is attractive to businesses that have an awesome team of marketing professionals on-board and are looking to improve their accounts performance through a few hourly sessions. Depending on the nature of the accounts and the skill-level of the businesses team members we sometimes recommend our Digital Marketing and Business Development Course as a more thorough investment of time. Below we've answered some of the most common questions we receive about our hourly consulting services. If we haven't answered your question below, please email us and we'll get back to you within 24 hours.
How many individual consulting sessions does it usually take to feel a tangible impact on marketing channels performance?
Of course, this varies from business to business. Most businesses feel a tangible impact in the performance of their campaigns within the first week. It truly depends on the level of digital infrastructure that has already been built for the business. If there is no digital infrastructure developed – it can take longer to plan and build. However, if it is just a matter of restructuring an existing digital strategy with digital properties already in existence, an impact can be felt after the first class.
What does an individual consulting session look like?
An individual consulting session is less structured than the full course and is ideal for businesses that need assistance with a specific component of their digital strategy. An individual consulting session lasts at minimum 2 hours and can be conducted in your office space or through any video/audio conferencing software. For pricing and further details please fill out the form below.
How much do you charge for hourly consulting?
Rates range depending on the nature of the help required and the skill-level of existing team members. Please fill out the form below explaining your situation and we'll get back to you with a quote within 24 hours.
Do you only consult for local businesses?
Not at all. We have consulted for businesses around the globe through screen sharing and web conferencing software like Skype and JoinMe. When it suits the nature of the business we're also happy to travel internationally to the businesses office for in-house consulting, additional fees will be negotiated.
What services do you offer online consulting for?
We can consult for any issues or accounts pertaining to Digital Marketing. Here are some of the most common services we consult on:
- Advertising (Google Adwords, Bing, Facebook, LinkedIn, Twitter, Youtube, Instagram etc.)
- Search Engine Optimization
- Content Optimization
- Social Media Marketing
- Public Relations
- Crowdfunding Strategy
- Investor Relations
- Influencer Marketing
- Web Design and Development
Who will perform the hourly consulting?
Our lead consultant: Vanessa Pintea, facilitates all consulting services. Check out her LinkedIn to explore her qualifications.


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Basic first aid is essential as part of your preparedness plan. Click here to see the first aid products that THRIVE has to offer.
Whether you buy a ready made kit or make your own, make sure you are prepared. Taking some first aid classes is also a way to be better prepared.
Do you want more of a natural first aid kit? doTerra has several options. Click here to check them out and see which one you would work for your family.
Kay Curtiss
Email: email@example.com
Let's Connect on Facebook: Thrive With Kay


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Ramacham Soap 100G
- Brand: The Origin Natural & Pure
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- Availability: Available
Chrysopogon zizanioides, commonly known as vetiver is a perennial bunchgrass of the Poaceae family, native to India.
The Richness Of Vetivert In The Form Of Extract And Oil Added In Coconut Oil Base Gives A Good Natural Deodorant.
Ingredients:- Coconut Oil, Virgin Coconut Oil, Castor Oil Ramacham Oil Glycerine, Caustic Lye, Herbal Extracts, Moisturiser, Perfume


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Mortimer Zuckerman on How We Can End Our Modern-Day Depression in US News: "We must increase investment in education and infrastructure, and enact tax reform":
If you went out this morning and saw hundreds of people lining up at soup kitchens, you'd think you were in a time machine, transported back to the Great Depression of the '30s, or the victim of a hallucination incubated by powerful images of those times. In fact, it is the absence of such images that is the illusion. We believe we live in more normal times—and we do not. Millions of people today are experiencing exactly the same struggle as the millions did in the Great Depression. They can't find work. They depend on government and philanthropy. They live on hope denied.
The big difference: Today millions are assisted by checks from Social Security and by food stamps. Food-stamp enrollment has been rising at the rate of 400,000 per month. More than 47 million Americans now depend on that program, an almost incredible record, for it is 15 percent of the population compared with the 7.9 percent who received food stamps from 1970 to 2000. Meanwhile, nearly 11 million Americans are now collecting federal disability checks from Social Security, and half have signed on since President Obama came to office. In 1992, there was one person on disability for every 35 workers. Today it is one for every 16. Such an increase simply cannot have been caused by direct disability experienced during employment. This is in effect another unemployment program, one without end. Many of the people on disability would normally be considered unemployed.
The reality is, we are experiencing a modern-day Depression. It is harder to find work than it has been in any previous economic recovery period. Typically, it takes 25 months to close the employment gap from the employment peak near the start of a downturn. But this time around, five years after employment peaked in January 2008, non-farm employment is still roughly 4 million below where it started. Never before has the job level not been at a record high during the fourth year into a business cycle.
In fact, 4.5 million fewer Americans are working today than when the recession started, and fewer are working today than in the year 2000, despite the fact that our population has grown by 31 million and our labor force by 11.4 million. Though the White House forecast four years ago that, with its stimulus policies, the jobless rate would be down to 5.2 percent by now, the real unemployment rate is 14.4 percent.
The Pew Research Center reports that for the first time in the post-World War II era, middle-class families finished the decade significantly poorer in terms of household net worth—which is down almost 40 percent since 2007—and with lower incomes than a decade earlier. This has hit the middle class harder than any other group. According to Pew, one third of Americans now identify themselves as lower class or lower middle class, a deterioration since 2008 when one quarter identified themselves that way.
The outlook is no better. Job seekers are only one third as likely to find a job now as they were in 2006. A record number of households have at least one member looking for a job. Some 40 percent of the people who are unemployed have been out of work for 27 weeks or more, which means we've got 4.8 million who are described as "long-term" unemployed. Employers are shortening the work week or asking employees to take unpaid leave in unprecedented numbers. Those taking unpaid leave are not included in the unemployment count.
Indeed, the recession has shown employers that they can make do with fewer workers. Now over 20 percent of companies say that employment in their firms will not return to pre-recession levels.
Older Americans, whose net worth has taken a huge hit (75 percent of it due to a decline in their home equity), can't afford to quit. Since the recession began, employment in that age group of 55 and older is up by over 3 million, in contrast to total employment, which is down by 5 million. The baby boomers are quite simply postponing their exit from the workforce. This has created a huge bottleneck for the nation's youth, who now face double-digit unemployment.


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I'm a cheater
I've been with my man for 6 years, and for most of the time we've been together i've slept with other men. It's not because i don't love him, because honestly i love him with all my heart. I just don't know why i feel the need to be involved with other men. I guess something is wrong with me. I want to come clean to him and start over because lately it is tearing me apart, but i know he would never forgive me. i don't know what to do.
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You will most certainly break your relationship if you do it. The thing is, you don't love him. Because a relationship implies compromises/sacrifices from both sides. You have to work on it, you know? It does not come easily or naturally. You think you're the only one who's been in a relationship and has been tempted by affairs? Temptations come from everywhere.
Anyhow, you've mentioned more partners. Whoops, that means that you're not really the type of girl who likes commitments or anything stable. You probably like experimenting. That's ok, I myself was built this way too and on top of that, I love receiving attention (even though it is sometimes focused on my physical aspect). However, knowing myself all too well, I reject any relationships. And what is wrong with you is that you are too selfish and want to have the best of both worlds: someone to come home to and someone to mess around with, without any implications.
You wanna have multiple partners and have a sense of independence from a relationship point of view? There's nothing wrong with that. BUT DON'T EVER mess with other people's feelings/intentions.
Although it's not very morale, if you really feel that bad and don't want to ruin the relationship officially, I don't recommend you telling him about your little adventures. It will be devastating for both of you. I'd suggest you immediately stop seeing other people. Next time he'll take you somewhere out or surprise you, you'll feel bad enough, so this kind of serves half of the punishment. Make it up for him, change yourself for the better. Wait like a dozen of years before you tell him.
This is the first option. If you don't feel like changing, then I suggest you leave him and tell him the truth. Accept the fact that you're not a relationships person and leave it. Just don't hurt other people.
Tell him; free yourself from guilt. Guilt can kill. Fast and pray and obtain forgiveness from God. Study God's word; so liberating.
If youd love him you wouldnt do things that would hurt him.. you're also a s***
You are evil! How could you say you love someone, but with your actions show the complete opposite! I hope he finds out and leaves you for someone who isnt a w****!
Tell him, or one day he will find out! Either start a fresh, with a solid foundation. Or walk away and stop hurting him, even though he doesnt know you are hurting him!
Don't tell him and stop cheating.


text:
FORTY-FIVE days is too short a time for a new government to show results, new Indian Finance Minister Arun Jaitley has said. Yet, his government's taking office under Prime Minister Narendra Modi after a decisive mandate in a contentious election needs watching.
It has signalled some of its political and economic priorities in an economic survey, a massive railway budget, the annual budget that Jaitley presented and much else.
Taking "much else" first, in his reply to President Pranab Mukherjee's speech opening the new Parliament, Modi cited three thinkers of contemporary India — Mahatma Gandhi, socialist leader Rammanohar Lohia and Deendayal Upadhyaya, one of the principal ideologues of the Bharatiya Janata Party — each emphasising that India cannot develop without developing its villages. If it were to happen, the said development would be incomplete.
This homegrown message is very different from what one has heard over the last half a century, or the last two decades when economic reforms and globalisation began. If this provides a philosophical grounding for Modi, who dislikes any "ism", and his government, there is a long-term solution for the country.
One would neither put complete hope on this course, nor discard it as Utopia. Now, moving to the nuts and bolts of governance, Modi has bemoaned the absence of a "honeymoon" for his new government. But it is enjoying one, mainly because shorn of rhetoric in and outside Parliament, and some negative developments that cannot be directly attributed to his government per se, it has, so far, been cautious.
And shorn of rhetoric again, the targets it has set for itself are modest. Both its budgets have been fairly innovative, underscoring thrust areas both old and new. But the basic arithmetic is the same as the pre-polls interim budget that the Manmohan Singh government had presented in February. This is because despite the changed political scenario, the economic problems confronting the country remain unchanged and demand a measure of continuity. Delivering on election promises would have to be gradual.
Commenting on the annual budget (2014-15) in Times of India, Swaminathan Aiyar called it (predecessor Congress finance minister) "P. Chidambaram's budget with saffron lipstick".
Halfway through Jaitley's budget speech, the Sensex plunged 300 points and recovered only after he proposed personal tax concessions for the middle-class, putting more money into its wallet and duty reduction for numerous goodies this class consumes.
He has also made the trade and industry sector happy. Business leaders are unanimous in welcoming the twin budgets because of the heavy investment in expanding and modernising infrastructure.
Now, India Inc. wants more and would be getting it. Their hope for a wider scope is laced with a challenge with foreign direct investment (FDI) mooted in the railways and hiked in defence and insurance sectors from 26 to 49 per cent. The FDI would come with the happy rider of full Indian management control.
Frankly, none of these are new. The question is who will deliver it and how, and who will get the credit. Congress, now in the opposition, charges the Modi government with "copying" its agenda, even as the latter says it inherited a bad economy.
Both are right — and both will continue to battle. This is certain, going by their respective track record of scoring political brownies on economic and social issues. For instance, the opposition is raising alarm over FDI in railway, knowing well that one of the world's largest rail network that is also the country's largest employer, needs serious revamping.
Of course, Jaitley's budgeting is an interim exercise. The next one should come within 10 months and offer a lot more scope for the government to be clear about its strategies, and allow it to be bold and decisive. The "hard" decisions, the unpopular ones, would come only next February.
For now, strategies for achieving them are missing. There are only indications of many bold strides on the intent to deliver on the election promises. Yet, keeping his poll promises, he has begun to deliver on smart cities, affordable housing, healthcare benefits, tax relief and incentives on savings.
The success of Modi's politics has been in cleverly combining the material aspirations of India's emergent middle-class, with the core ideology of the Sangh Parivar, the Hindu rightwing "nationalist" platform, as a subtext. Jaitley followed that formula. And this is more significant than the economics of the budget.
By naming new schemes after the ruling BJP's ideological forebears — Syama Prasad Mookerjee, Deendayal Upadhyaya and Madan Mohan Malaviya — Jaitley gave reassuring signals to Hindutva, the core political constituency.
Calling for an "intellectual manthan (churning)", Modi said Indians should move beyond roads, highways and ports — "New cities will be built where there are I-ways, not only highways".
His thoughts found echoes in Jaitley's speech.
"There is an imminent need to further bridge the divide between digital haves and have-nots," he said while announcing a programme called Digital India to ensure broadband connectivity at the village level and improved access to services through IT-enabled platforms.
Urbanisation and connectivity are natural corollaries. The rail budget promises Wi-Fi services in larger stations and in select trains. Free Wi-Fi is coming at airports and most coffeeshops. India is now the world's third largest Internet user after China and the United States. Everyman is sporting smartphones for bank transactions, bill payments, catching up on course work, booking holidays, hooking up with friends, business communication, having fun and doing work.
Though similar schemes were announced and initiated by the previous Manmohan Singh government, too, the BJP finds the political potential in it. This, again, underscores the need for continuity with cautious thrusts into the new areas. There are no short cuts to development. The urban India and the rural Bharat need to meet halfway.
The manthan that Modi advocates, would need to be sustained, with its course carefully stayed, for India to achieve its immediate and long-term goals.