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Although long noncoding RNAs (lncRNAs) are proposed to play essential roles in mammalian neurodevelopment, we know little of their functions from their disruption in vivo. Combining evidence for evolutionary constraint and conserved expression data, we previously identified candidate lncRNAs that might play important and conserved roles in brain function. Here, we demonstrate that the sequence and neuronal transcription of lncRNAs transcribed from the previously uncharacterized Visc locus are conserved across diverse mammals. Consequently, one of these lncRNAs, Visc-2, was selected for targeted deletion in the mouse, and knockout animals were subjected to an extremely detailed anatomical and behavioral characterization. Despite a neurodevelopmental expression pattern of Visc-2 that is highly localized to the cortex and sites of neurogenesis, anomalies in neither cytoarchitecture nor neuroproliferation were identified in knockout mice. In addition, no abnormal motor, sensory, anxiety, or cognitive behavioral phenotypes were observed. These results are important because they contribute to a growing body of evidence that lncRNA loci contribute on average far less to brain and biological functions than protein-coding loci. A high-throughput knockout program focussing on lncRNAs, similar to that currently underway for protein-coding genes, will be required to establish the distribution of their organismal functions.
The full range and complexity of RNA species that are transcribed in mammals are becoming increasingly apparent as a result of large-scale cDNA cloning and transcriptome sequencing projects (Okazaki et al. 2002; Carninci et al. 2005; Birney et al. 2007; Derrien et al. 2012). A substantial proportion of these transcripts are not translated: in addition to a myriad of small regulatory RNAs such as microRNAs (miRNAs), many thousands of long noncoding RNAs (lncRNAs) have been identified (Guttman et al. 2009; Derrien et al. 2012). These larger transcripts, defined as being over 200 nucleotides in length, are found not only within, or anti-sense to, protein-coding genes, but also in intergenic regions (Khalil et al. 2009). Despite the prediction that the number of distinct lncRNAs expressed in humans may surpass that of protein-coding genes (Derrien et al. 2012), very few of these have been studied experimentally in any detail and the molecular functions of only a handful have been described to date (Moran et al. 2012).
In the absence of abundant mechanistic findings, comparative evolutionary methods have been employed to show that evolutionary constraint within lncRNAs is generally low compared with protein-coding genes, even for closely related species (Ponjavic et al. 2007; Marques and Ponting 2009). For example, it has been estimated that 81% of human lncRNAs are confined to primates (Necsulea et al. 2014). However, some examples of conserved expression and functionality have begun to emerge (Chodroff et al. 2010; Ulitsky et al. 2011). In vitro studies, predominantly based on manipulating the expression levels of individual lncRNAs, have yielded some mechanistic insights and implicate these transcripts in diverse cellular processes (Wang and Chang 2011). Some lncRNA loci coincide with enhancer marks and their transcription is associated with modest increases in transcript abundance for neighboring protein-coding genes (Kim et al. 2010). Other lncRNAs act in trans via their interactions with intermediate nucleic acids or proteins (Moran et al. 2012; Vance et al. 2014). Potential functional significance has also been inferred from expression patterns of lncRNAs; for example, recent next-generation sequencing studies of mRNAs in the mammalian cortex have identified transcripts with a layer-specific distribution (Belgard et al. 2011).
To resolve the issue of functionality, it will be critical not just to consider lncRNA cellular roles and molecular mechanisms in the small set of chosen cell types that are assayed, but also to reveal their organismal functions by targeted disruption of their loci in model organisms. Only a relatively small number of mouse lncRNA knockout models have been reported thus far. For example, disruption of Evf2 results in a subtle neuronal cellular phenotype (Bond et al. 2009) and loss of Dlx1as, the anti-sense lncRNA to transcription factor Dlx1, results in mild neurological defects (Kraus et al. 2013). Of 18 lncRNA knockout lines recently described, 5 were reported to have observable phenotypes including Fendrr, whose disruption was previously reported to result in embryonic lethality (Grote et al. 2013; Sauvageau et al. 2013). Reproducible cellular phenotypes have been observed in vitro by manipulation of Neat1, Malat1, and HOTAIR lncRNAs; however, no detectable physiological abnormalities were discernible in their corresponding constitutive knockout mouse models (Nakagawa et al. 2011, 2012; Schorderet and Duboule 2011; Eissmann et al. 2012; Zhang et al. 2012). In the case of Malat1, this was particularly unexpected given this transcript's exceptionally high level of expression reported in many tissues, including the brain, as well as evidence for a role of its lncRNA in pre-mRNA splicing recruitment and even cancer progression (Tripathi et al. 2010; Gutschner et al. 2013).
Without many further lncRNA knockout models, it would be premature to draw general conclusions from experimental data regarding the functional importance of lncRNA loci relative to protein-coding genes in mammalian systems. In addition, few studies have investigated the effects of targeted deletion of specific transcripts, while preserving other transcripts, in complex noncoding loci (Kraus et al. 2013). In this study, we investigated the phenotypic effect of disrupting a mouse lncRNA, Visc-2, from just such a complex locus. This transcript was chosen as it has been conserved in both sequence and brain expression across diverse mammals. We anticipated from its expression profile that effects of this disruption might be manifested in altered brain anatomy and/or behavior.
Materials and Methods
Multispecies Sequence Alignments
Regions orthologous to Visc-1 and Visc-2 (including the flanking 100 kb on each side of the indicated locus as shown below) were obtained from the following whole-genome sequence assemblies (available from the UCSC Genome Browser at http://genome.ucsc.edu): frog (Xenopus tropicalis; xenTro2), chicken (Gallus gallus; galGal3), songbird (Taeniopygia guttata; taeGut1), lizard (Anolis carolinensis; anoCar1), platypus (Ornithorhyncus anatinus; ornAna1), opossum (Monodelphis domestica; monDom4), mouse (Mus musculus; Mm9), rat (Rattus norvegicus; Rn4), guinea pig (Cavia porcellus; cavPor3), marmoset (Callithrix jacchus; calJac1), macaque (Macaca mulatta; rheMac2), orangutan (Pongo abelli; ponAbe2), human (Homo sapiens; Hg18), chimpanzee (Pan troglodytes; panTro2), horse (Equus caballus; equCab1), dog (Canis familiaris; canFam2), and cattle (Bos taurus; bosTau3). The liftOver program (Kent et al. 2003) was used to identify singly mapped (unambiguous) orthologous regions in the nonmouse species genomes. TBA (Threaded Blockset Aligner) was used to generate multisequence alignments (Blanchette et al. 2004) and visualized with Gmaj (Generalized Multiple Alignments with Java; Blanchette et al. 2004). Sequence identity plots were graphed with the program SinicView (Sequence-aligning INnovative and Interactive Comparison VIEWer; Shih et al. 2006). The corresponding genomic co-ordinates were selected: A. carolinensis scaffold_184:178408-1648757; B. taurus chr7:88511771-90384894; C. jacchus Contig1239:4-413851; C. jacchus Contig2424:1164-306723; C. jacchus Contig5:723588-1953659; C. familiaris chr3:22038316-23682511; C. porcellus scaffold_1:15464040-17198010; E. caballus chr14:78013389-79614242; G. gallus chrZ:58785269-59599138; H. sapiens chr5:86962138-88980753; M. mulatta chr6:83943471-85955231; M. domestica chr3:214376700-216926449; M. musculus chr13:82803274-85042441; O. anatinus chr1:5730158-7289157; P. troglodytes chr5:25947637-28003787; Pongo abelii chr5:88205383-90270057; R. norvegicus chr2:11050426-13768375; T. guttata chrZ:11967213-12578404; X. tropicalis scaffold_76:2296352-2888843.
In Situ Hybridization
Target sequences were generated by PCR or reverse transcriptase (RT)–PCR, cloned into pCR4-TOPO (Invitrogen) and digoxigenin-labeled riboprobes were synthesized from linearized plasmid DNA. Tissue samples were snap frozen in OCT and 14 μm sections were cut using a cryostat (Leica) and mounted onto Superfrost Plus slides (VWR). Frozen sections of female human fetal frontal cortex (35 weeks gestation) were cut at 4 μm (Amsbio). Probe hybridization, washing, and signal detection using an alkaline phosphatase-conjugated anti-DIG antibody was carried out as previously described (Chodroff et al. 2010). Primer sequences for riboprobe cloning: Visc-2 F 5′ GGATCACCATGACTGATCAG and Visc-2 R 5′ ACCATTCTACCACTTGAGCC; Visc-1 F 5′ GTTCACATCTCCTGCTTTCC and Visc-1 R 5′ CCTGGAAACTTTCTATGTATT; AK036048 F 5′ CTTCCTCGTAGAACACTGGAG and AK036048 R 5′ CACCACACAGATTGCTAAGTG; Visc-3 F 5′ CCTTATGCATTCTGCATTCATG and Visc-3 R 5′ CTAGCTGAGCTACAGAATATC; Human AK125167 F 5′ GTTTCGCGTGTTGTCAGCTG and AK125167 R 5′ ATGGTACGAATATGGCTTCTTTC; Opossum Visc-2 orthologue F 5′ YTATTGGATYACYATGTCTGATCAGCTG and 5′ CCAGTCTCYTCYAGGTMSTCACC.
Quantitative RT–PCR and RACE PCR
qPCR analyses of Visc-1, Mef2c, Tmem161b, Visc-3, and AK082072 were performed on a StepOne plus (ABI) using Power-SYBR master mix in a total volume of 20 μL. Measured fluorescence was normalized against GAPDH. Total RNA was extracted from whole brains using the RNeasy Mini kit (Qiagen) and 100 ng was reversed transcribed using Superscript II (Invitrogen) as per the manufacturer's instructions. Primers used were: Visc-1 F 5′ GGACAACACTGCCTGGCGCAG and Visc-1 R 5′ GGACAACACTGCCTGGCGCAGAC; Visc-3 F 5′ CTGAGTCTTGCCTCGAGTATCTGTGC and Visc-3 R 5′ GTGTCATTTGATCTGTCTGCGCCAGGC; Mef2c F 5′ CACGATGC CATCAGTGAATCAAAGG and Mef2c R 5′ GCCGAGGTGGAGCGCACTGGCAGTG; Tmem161b F 5′ GTTCTTGGCGTTATGGGTGTGATAGG and Tmem161b R 5′ TGCTTCCCTGCAAGGATTCTTAATTC; AK082072 F 5′ GTCAATAAAGGAATGTCCATCTG and AK082072 R 5′ CTAGAATTGGCATGGCATCACTC; Gapdh F 5′ TGTGTCCGTCGTGGATCTGA and Gapdh R 5′ CCTGCTTCACCACCTTCTTGA. All primers were assessed for efficiencies of the target and reference gene being approximately equal (absolute value of the log of the slope being <0.1). Four age- and sex-matched animals from each genotype were used and all reactions were performed in triplicate and analyzed using the comparative Ct method. Quantitative PCR analysis of miR-9-2 was carried out using cDNA generated by the Taqman MicroRNA Reverse Transcription Kit (ABI). The Taqman Universal PCR Master Mix (ABI) was used with the snoRNA234 Taqman Small RNA Assays kit (ABI) as the reference, and data were analyzed using the comparative Ct method. 5′ and 3′ rapid amplification of cDNA ends (RACE) reactions were carried out using the RLM RACE system (Invitrogen) according to the manufacturer's instructions. PCR products from nested RT–PCR were gel-purified prior to sequencing.
Generation of Visc-2−/− Mice
pL452 and pL253 vectors were supplied by the Biological Resources Branch, NCI-Frederick Cancer Research, and Development Center (Liu et al. 2003). To insert components of pL452 into the bacterial artificial chromosome (BAC), the vector was first manipulated to include regions flanking the 5′ and 3′ ends of Visc-2 by PCR. An approximately 800-bp fragment immediately upstream of Visc-2 was cloned into pL452 upstream of the loxP site and PGK promoter. An approximately 800-bp fragment immediately downstream of Visc-2 and within the first intron of Visc-1 was also cloned directly downstream of bGHpA and a loxP site in pL452. The resulting pL452 components flanked by the 5′ and 3′ arms were removed by restriction digest and transformed into induced EL350 cells containing the RP23-451C5 BAC; incorporation of pL452 components into the BAC was confirmed by restriction enzyme digestion and sequencing (now termed BAC RP23-451C5-A). The targeting vector was also generated by recombineering using a modified pL253 vector containing homologous segments of RP23-451C5 flanking the region of interest. The required fragment was removed from pL253 by restriction digest and transformed into induced EL350 cells containing BAC RP23-451C5-A. The modified BAC was verified by restriction enzyme digestion and sequencing. Linearized DNA from this construct was transfected into ES cells by electroporation and screening for positive clones was carried out by Southern blotting of ScaI-digested genomic DNA. Eight-cell aggregations were carried out using embryos removed from pregnant BALB/c female mice and expanded C57BL/6J-derived, GFP-expressing mouse ES (LC3) cells; thus chimeric offspring could be identified by coat color or GFP expression. Chimeric mice were crossed with C57BL/6J wild-type mice, and transgene integration was confirmed by PCR using the primers WT_F (for the wild-type allele) 5′ AAAGAGCCCAGTCCCAATCT; HET_F (for the mutant allele) 5′ TCGACTAGAGCTTGCGGAAC and HET_WT_R (for wild-type and mutant) 5′ TGTCACCCCCAAATCATAGC.
All mice used in this study were derived from Visc-2+/− × Visc-2+/− matings. Before dissection or behavioral testing, the mice were housed on a 12/12-h light/dark cycle (lights on at 0700 h and off at 1900 h). Mice were housed in groups of 4–5 with ad libitum food and water. All testing was performed during the light phase. The maintenance and testing of these animals were performed under the UK Home Office guidelines for the treatment of animals under scientific procedures and the local ethical review board at the University of Oxford.
Neuroanatomical Screening
Adult (8 weeks of age [P56]) mice were perfused transcardially with 0.1 M PBS, followed by 4% paraformaldehyde (PFA). Brains were dissected out and post-fixed in 4% PFA at 4 °C overnight, then transferred to 30% sucrose for cryoprotection. Brains taken at P1 were dissected and immersion fixed in 4% PFA for 48 h prior to cryoprotection. Brains were cut at 30 μm using a freezing microtome (Leica) or at 20 μm using a cryostat (Bright). For Nissl staining (N = 3 Visc-2+/+ and N = 3 Visc-2−/− at P56), every 12th section along the rostrocaudal axis was selected. For cortical layer marker studies (N = 3 Visc-2+/+ and N = 3 Visc-2−/− at P1 and P56, respectively), equivalent regions of the somatosensory cortex were mounted on slides and blocked in 5% donkey or goat serum in PBS with 0.3% Triton X-100. Primary antibodies were incubated in the same blocking solution overnight at 4 °C: Nurr1 (1 : 50, R&D Systems), Foxp2 (1 : 1000, Abcam), Cux1 (1 : 50, Santa Cruz), Satb2 (1 : 100, Abcam), Tbr1 (1 : 400 Abcam), and Ctip2 (1 : 800 Abcam). Immunostraining for Nurr1, Tbr1, Satb2, and Ctip2 required additional retrieval in 10 mM sodium citrate pH 6.0 buffer (20 min) prior to blocking. The appropriate secondary antibodies (Alexa-488, Invitrogen) were applied for 2 h at room temperature.
For OB and rostral migratory stream (RMS) immunohistochemistry (N = 4 Visc-2+/+ and N = 5 Visc-2−/−), sections were counterstained with DAPI (5 μg/mL, Invitrogen) for 15 min (to allow confirmation of rostrocaudal levels), and blocked in 10% donkey or goat serum in PBS with 0.1% Triton X-100. Primary antibodies were incubated in the same blocking solution and incubated overnight at 4 °C: phosphohistone H3 (Ser10; pHH3; 1 : 500, Millipore), doublecortin (Dcx; 1 : 100, Millipore), glial fibrillary acidic protein (GFAP; 1 : 500, DAKO), calbindin D-28-K (CB; 1 : 2000, Swant), and calretinin (CR; 1 : 2000, Millipore). Secondary antibodies were applied for 90 min at room temperature: either donkey or goat anti-rabbit IgG Alexa-568 or -546 (Invitrogen), or for Dcx immunostaining, an additional biotin–streptavidin amplification (using biotinylated donkey anti-goat IgG [Abcam] and streptavidin Cy5 [USBiologicals]) was applied. To assess the subventricular zone (SVZ), 4 sections through levels within the range bregma 1.10–0.38 mm, interaural 4.90–4.18 mm (8 lateral ventricles in total per brain), were stained by immunohistochemistry. For quantification, the number of pHH3+ cells in the SVZ surrounding each lateral ventricle was counted. Note that pHH3 staining pattern depends on the phosphorylation state of the histone H3 protein, which changes during the cell cycle; only cells with uniformly intense nuclear staining (that reaches a maximum at metaphase) were included in the analyses. For assessment of the OB, 2 hemispheric sections from the mid-region of the rostrocaudal axis of the OB, and 2 from the more caudal OB, were used per brain. The number of CB+ cells in the glomerular layer was quantified by positioning four 200 μm × 200 μm boxes in this layer within each section, using Photoshop CS6. The same procedure was applied for CR+ cells; however, in addition 4 boxes were positioned in the granule cell layer of each section. For RMS analysis, 4 equivalent representative sections were taken from each brain and immunostained for pHH3 and Dcx.
For newborn neuron quantification in the OB, bromodeoxyuridine (BrdU) was injected intraperitoneally (i.p.) at 50 mg/kg once daily at P7 for 6 days (N = 4 Visc-2+/+ and N = 4 Visc-2−/−). After 14 days, the animals were perfused with 4% PFA, post-fixed in 4% PFA overnight, and cryprotected before cutting at 20 μm. Five equivalent coronal sections through the OB were first subjected to antigen retrieval (20 min in 10 mM sodium citrate pH 6) followed by water and PBS washes before blocking in 5% donkey serum with 0.3% Triton X-100. The sections were then incubated with sheep anti-BrdU (1 : 500, Abcam) made in the same blocking buffer overnight at 4 °C. After washing in PBS, donkey anti-sheep Alexa-488 was applied for 2 h. Co-staining for NeuN was carried out after additional blocking in 5% goat serum with 0.3% Triton X-100 using mouse anti-NeuN (1 : 500, Millipore) made in the same blocking buffer overnight at 4 °C. After washing in PBS, goat anti-mouse Alexa-594 was applied for 2 h before counterstaining with DAPI and mounting. For live counting of BrdU-positive (BrdU+) and BrdU/NeuN double-positive (BrdU+/NeuN+) cells, 5 fields of view at ×20 magnification were counted from both hemispheres from each of 5 equivalent sections from 4 mice of each genotype. All image quantification was performed blindly.
Carbocyanine Dye Tracing
P7 mice were perfused with 4% PFA as above, the brains were dissected out and post-fixed in 4% PFA for 24 h, followed by long-term storage in 0.1 M PBS with 0.05% sodium azide. The brains were bisected along the coronal plane at the level of the posterior thalamus, so the cut surface contained ventrobasal complex, internal capsule, and the cerebral peduncle. Small crystals of the carbocyanine dye DiI (1,1′-didodecyl-3,3,3′,3′-tetra-methylindocarbocyanine perchlorate [(Invitrogen]) were placed under direct visual guidance, using a tungsten wire, into the cut surface of the thalamus (right hemisphere) and internal capsule (left hemisphere). We selected relatively larger crystals (up to ≈60 μm) for DiI placements into the cerebral peduncle and ventrobasal complex to achieve bulk labeling. Brains were incubated at 37 °C for 6 weeks according to previous protocols, to provide sufficient time for the diffusion of the dye within the cell membrane and accomplish both anterograde and retrograde labeling (Molnár et al. 2006). Following the incubation, the brains were embedded in 5% agarose (Bioline) and sectioned into 100 μm coronal sections on a vibrating microtome (VT1000S; Leica, Germany). They were counterstained with bisbenzimide (2.5 μg/100 mL, Hoechst 33258; Sigma) and mounted using 0.1 M phosphate buffer (PB) before being viewed on an upright epifluorescence microscope (Leica).
Behavioral Testing
All behavioral testing was carried out on a cohort of 10-week-old male Visc-2−/− and Visc-2+/+ (N = 12 for each genotype for each assay). Testing was carried out over a period of 4 weeks in the order described below.
Locomotor Activity
To measure novelty induced locomotor activity (LMA), experiments were conducted in the light phase between 1000 and 1400 h. Ambulations were measured in transparent plexiglass cages (20 × 35 cm), equipped with infrared photobeams (San Diego Instruments) over a total of 4 h.
Elevated Plus Maze (EPM)
The elevated plus maze (EPM) consisted of arms 30 cm long and 5 cm wide with the 2 opposing closed arms with 30 cm sides. Mice were placed in the EPM facing the open arm and allowed to freely explore the apparatus over 5-min trial using the Ethovision tracking software (Noldus). Entry into an arm of the EPM was defined using the base of the tail as the tracking position. The percentage spent exploring the open arms was calculated by dividing the time spent in the open arms by the combined time spent in open and closed arms.
Anxiogenic Open Field
Behavior was assessed in a white, anxiogenic open field (60 cm diameter, lighting at 3000 lux). An Ethovision software (Noldus) was used to split the arena into a central area of 10 cm radius and an outer area of 20 cm radius. Mice were placed into the apparatus at the edge and the distribution of time spent in the central and peripheral areas was taken as a measure of anxiety. The total distance traveled was also recorded.
Spontaneous Alternation in a T-maze
The apparatus consisted of a black T-shaped wooden maze made of arms, measuring 30 cm in length, 10 cm in width, and 29 cm in height. The goal arm entrances were provided with sliding guillotine doors. A central partition wall, extending 7 cm into the start arm, divided the choice point into 2 goal arms to prevent the mouse from receiving any sensory input from the nonvisited arm. A mouse was placed in the start arm of the T-maze and allowed to choose a goal arm. The mouse was then confined to the goal arm by sliding the guillotine door down. The arm entered (left or right) was recorded and the mouse was allowed to explore the goal arm for 30 s. The mouse was returned to the start arm, with the guillotine doors re-opened and the central partition removed, and allowed to explore again. The goal arm entered on the second run was recorded (left or right) and the mouse was then returned to its home cage. Each mouse was tested twice each day for 5 days (a total of 10 trials). The percentage of trials in which the mouse entered a different goal arm on the second run (i.e., alternated) was calculated.
Olfactory Preference Test
As a measure of the sense of smell olfactory response to both attractive and aversive scents was assessed as previously described (Witt et al. 2009).
Spatial Novelty Preference in the Y-Maze
Spatial novelty preference was assessed in an enclosed Perspex Y-maze with arms of 30 × 8 × 20 cm placed into a room containing a variety of extramaze cues. Mice were assigned 2 arms (the "start" and the "other" arm) to which they were exposed during the first phase (the exposure phase), for 5 min. This selection of arms was counterbalanced with respect to the genotype. Timing of the 5-min period began only once the mouse had left the start arm. The mouse was then removed from the maze and returned to its homecage for a 1-min interval between the exposure and test phases. During the test phase, mice were allowed free access to all 3 arms. Mice were placed at the end of the start arm and allowed to explore all 3 arms for 2 min beginning once they had left the start arm. An entry into an arm was defined by a mouse placing all 4 paws inside an arm. Similarly, a mouse was considered to have left an arm if all 4 paws were placed outside the arm. The times that mice spent in each arm were recorded manually and a novelty preference ratio was calculated for the time spent in arms [novel arm/(novel + other arm)].
Spatial Reference Memory: Morris Watermaze
The watermaze (diameter 2.0 m) was filled with water to a depth of 45 cm containing 500 mL of white poster paint to render the water opaque. In order to escape from the water the mice had to find a fixed location, hidden escape platform (diameter 21 cm) submerged approximately 1 cm below the water surface. The platform was located at the center of one of opposite quadrants (arbitrarily designated NW and SE). The number of mice trained to each platform position was counterbalanced with respect to the group. Animals received 4 trials per day for 9 days, with an intertrial interval of approximately 15 s. Mice were placed into the pool facing the side wall at 1 of 8 possible start locations (nominally N, S, E, W, NE, NW, SE, and SW, chosen randomly across trials), and allowed to swim until they found the platform, or for a maximum of 90 s. Any mouse that failed to find the platform within the allotted time was guided to the platform. The animal then remained on the platform for 30 s before commencing the next trial. The time taken to locate the platform and the distance traveled was quantified using the Anymaze (Stoelting) software. On the seventh (24 h after spatial training trial number 24) and tenth days of testing (24 h after spatial training trial 36), a probe trial was conducted to determine the extent to which the mice had learned about the spatial location of the platform. The platform was removed from the pool, and the mice were allowed to swim freely for 60 s. The percentage of time that animals spent in each quadrant of the maze was recorded.
Genbank Submissions
BAC assemblies: spider monkey, DP001046; chicken, DP00104; echidna, DP001048; ferret, DP001049; gorilla, DP001050; guinea pig, DP001051; macaque, DP001052; marmoset, DP001053; opossum, DP001054; mouse lemur, DP001055; pig, DP001056; platypus, DP001057; zebrafinch, DP001058. RACE PCR of mouse Visc-1: KF032589 and KF032590.
We previously identified the sequence of a brain-expressed lncRNA (accession number AK158494) as being highly conserved among mammals (Ponjavic et al. 2007). This single-exon lncRNA was identified originally from mouse visual cortex and was found among a set of cDNAs that were enriched for full-length sequences (Okazaki et al. 2002). Its locus overlaps with expressed sequence tags from cDNA libraries derived from both visual cortex and retina. The lncRNA is transcribed from an intergenic region of mouse chromosome 13 between Mef2c and Tmem161b (Fig. 1A). The AK158494 locus partially overlaps with the first exon and intron of an Ensembl-predicted multi-exonic lncRNA ENSMUST00000131907 (Fig. 1B). We thus confirmed the expression and sequence limits of both these transcripts in the embryonic mouse brain using both RT–PCR and 5′/3′-RACE (KF032589 and KF032590). Each of these lncRNAs was readily detectable, with the spliced variant showing 4 different canonical GT-AG introns, and both transcripts sharing a single putative promoter. In addition, both transcripts contain Evofold-predicted RNA secondary structures (Fig. 1C), but neither lncRNA contains an open reading frame that exceeds 100 amino acids. We shall refer to the locus as Visc, chosen to reflect the known expression of expressed sequence tags (ESTs) in the region in the visual cortex, and to these 2 Visc lncRNAs as Visc-1 (multi-exonic; ENSMUST00000131907) and Visc-2 (single-exonic; AK158494).
DNA sequence of the Visc locus aligns with the genomes of evolutionarily distant vertebrates such as the frog X. tropicalis (Fig. 1C). In addition, alignment of the mouse Visc-1 multi-exonic sequence with multiple vertebrate genome sequences revealed that all donor and acceptor dinucleotide splice sites and a polyadenylation signal were conserved in evolution across mouse, human, and cattle, with only 2 substitutions in the metatherian opossum (M. domestica; Supplementary Fig. 1A). However, sequence conservation from mouse to opossum from the putative transcription start site (TSS) of mouse Visc-1/2 was relatively modest (up to 66% sequence identity over 0.2 kb) and its sequence failed to align to nonmammalian vertebrate genomes. We therefore expected to identify only eutherian and metatherian Visc-1 multi-exon orthologues, as well as Visc-2 single-exon variants, possibly originating from distinct TSSs. A Visc-2 orthologue was indeed identified in human (2.82 kb; AK125167), and as predicted, this transcript shares neither a TSS nor a termination site with its mouse counterpart, but it is 67% identical in sequence based on a local alignment (Fig. 1D). Sequence alignments also revealed human (3.4 kb; LOC645323), cattle (4 kb; EE241071), and pig (3.99 kb; CK461258) orthologues of Visc-1 transcripts with a conserved exonic structure and sequence identities of 82% over 3.4 kb, 80% over 0.63 kb, and 86% over 0.73 kb, respectively (Supplementary Fig. 1B). Significantly, the tissue specificity of transcription appears to be conserved among species because these homologous cDNAs have been isolated from nervous system tissue (Supplementary Table 1). It is also noteworthy that conservation in the terminal 3′ exon of Visc-1 is likely due, at least in part, to the presence of the micro (mi)RNA-9-2 (miR-9-2) precursor in its exonic sequence (Fig. 1C). Visc-1 can thus be considered to be an mRNA-like noncoding RNA (Rodriguez et al. 2004), which is a miRNA precursor transcript, pre-miRNA9-2, in contrast to Visc-2 which does not harbor miRNA sequences.
To characterize the tissue expression pattern of the Visc locus in more detail, in situ hybridization was carried out in the embryonic (E)16.5 mouse brain. Riboprobes were designed to investigate all of the annotated mouse lncRNAs in the region, including an additional transcript anti-sense (AK036048) and an additional splice variant, Visc-3 (AK039679), which contains an alternative 3′ terminal exon (Fig. 1B). These data revealed that both Visc-1 and Visc-2 are preferentially expressed in the developing forebrain and are co-localized with the rostral and caudal interneuron migratory streams in addition to the cortical plate and the ventricular and subventricular zones of the telencephalon (Fig. 1E). To investigate the temporal distribution of this highly specific expression pattern, additional Visc-2 in situ hybridization data were generated, showing that this lncRNA is expressed in the developing telencephalon at E13.5 but is not detectable outside of the brain (Fig. 2A). Furthermore, at early postnatal time points, expression is detected in all cortical layers, although it appears more pronounced in the neuroproliferative SVZ, RMS, and olfactory bulb (OB) as well as the granule cell layer of the cerebellum; this distribution continues into adulthood (Fig. 2B–D).
We have previously demonstrated that spatiotemporal expression of lncRNAs can be conserved in developing avian, marsupial, and eutherian brains (Chodroff et al. 2010). To determine whether there was similar evidence for Visc-2, expression of the orthologous opossum lncRNA was first confirmed by RT–PCR. Subsequently, in situ hybridization showed that expression of this lncRNA in the postnatal opossum brain occurs in neuroproliferative zones, including the telencephalic ventricular zone similar to that seen in the pre- and early postnatal mouse brain (Fig. 2E). We were, however, unable to identify a chicken orthologue for Visc-2. In addition, due to the apparent high level of sequence identity between mouse Visc-2 and its human orthologue AK125167 (Fig. 1D), we sought to confirm whether expression of the predicted human lncRNA was detectable by in situ hybridization. Fetal frontal lobe sections show the expression of AK125167 in all cortical layers at an estimated developmental time point that can be considered equivalent to the early postnatal stage in the mouse cerebral cortex (Fig. 2F; Clancy et al. 2001). Taken together, these data provide evidence for evolutionary conservation in sequence and in the regulation of transcription of Visc-2 across distantly related mammals, suggesting that this lncRNA plays an important and conserved functional role in the developing mammalian nervous system.
To gain greater insights into the organismal function of Visc-2 in vivo, a constitutive knockout mouse was generated. As the Visc-2 transcript shares a predicted TSS with Visc-1 (see above), our strategy to attenuate expression was to selectively delete the entire Visc-2 locus (including the Visc-1/Visc-2 TSS), with the expectation that transcription of both lncRNAs would be abrogated. A BAC clone spanning the first 2 exons of Visc-1 was modified by recombineering and subsequently subcloned to create a targeting construct (Fig. 3A,B). This construct was electroporated into embryonic stem (ES) cells and homologous recombination was confirmed by Southern blotting (Fig. 3C). Aggregation of ES cells heterozygous for the targeting construct into 8-cell embryos produced one viable and fertile male mouse with an estimated high percentage of chimerism. Subsequent breeding from this chimera generated the expected Mendelian inheritance ratios of wild-type (Visc-2+/+), heterozygous (Visc-2+/−), and knockout (Visc-2−/−) offspring of both sexes (Fig. 3D; Supplementary Table 2). The loss of the Visc-2 transcript was also confirmed by RT–PCR from the E16.5 brain (Fig. 3E). Initial examination of Visc-2−/− mice revealed that they are fertile and appear to develop normally, with neither male nor female knockout animals showing significant differences in size, weight, or lifespan relative to Visc-2+/− or Visc-2+/+ controls from birth to adulthood up to 18 months of age (data not shown).
To assess whether the loss of Visc-2 influenced the expression of genomically neighboring loci, qRT–PCR analysis of the E16.5 brain was performed to examine genes present in a large genomic region flanking the deleted locus (Figs 1A and 4A). These data show there to be no significant difference in the expression levels of flanking protein-coding genes Tmem161b or Mef2c, or a conserved lncRNA present over 200 kb downstream of Visc-2, AK082072 (Fig. 4B). Unexpectedly, there was no influence of the deletion of Visc-2 on the expression of Visc-1 as shown by qRT–PCR (Fig. 4B) and in situ hybridization (Fig. 4C,D), indicating that alternative Visc-1 RNAs are transcribed from additional promoters that are not shared with the deleted Visc-2 locus. Nevertheless, expression level of the miR-9-2 mRNA was significantly reduced by approximately 30% in knockout brain tissue at this time point (P = 0.032; Fig. 4B). Complete abrogation of expression of mRNA-9-2 in Visc-2−/− mice was not expected because expression of a major transcript in human neuroblastoma cells requires a promoter only just upstream of the pre-miRNA 5′ end (Laneve et al. 2010).
An initial overview of the gross morphology of Visc-2−/− brains by Nissl staining uncovered no abnormalities in structure or cytoarchitecture, compared with controls (Supplementary Fig. 2). Although no overt anatomical changes were observed, this warranted a more detailed regional study. Initially, considering the high levels of Visc-2 in the developing cortex, markers with known layer-specific patterns of expression in this region were analyzed at early postnatal (P1) and adult (P56) in Visc-2−/− and Visc-2+/+ mice (Supplementary Fig. 3). These data show normal distribution of markers in the subplate (Nurr1), through layers 4–6 (Ctip2, Foxp2, and Tbr1) to the upper layers 2–3 (Satb2 and Cux1) at both time points, suggesting that no overt patterning or migration defects occur in the cortex of Visc-2−/− mice.
In addition, because the expression of Visc-2 in the SVZ, RMS, and OB persists throughout postnatal development (Fig. 2), these 3 structures were examined in greater detail to determine whether neurogenesis within the adult mouse olfactory system was influenced by Visc-2 loss. Immunohistochemistry was used to quantitatively investigate the structural integrity of the RMS, generation of neuroblasts in the SVZ, as well as neuronal fate specification and distribution within the OB—the endpoint of the newly generated neurons migrating from the SVZ. The RMS itself failed to show any change in overall positioning, size, or structure between Visc-2+/+ and Visc-2−/− mice. The basic internal organization, with astrocyte cell processes surrounding immature migrating neuroblasts, was also unaltered as shown by the similar distribution and lack of co-localization between GFAP and Dcx immunostaining (Fig. 5A). Quantitative assessment of the SVZ was carried out using phosphohistone H3 (pHH3 as a marker for cell cycle progression; Ponti et al. 2013). In both genotypes, the vast majority of pHH3+ dividing cells were present along the lateral wall of the lateral ventricle (Fig. 5B). In addition, the distribution of Dcx-positive (Dcx+) migratory neuroblasts in this region (as well as in the corpus callosum and the striatum) was also very similar for animals of both genotypes (Fig. 5C). The size of the proliferative population indicated by pHH3 immunostaining was reduced in the Visc-2−/− SVZ relative to that of the Visc-2+/+ (Fig. 5E), although this failed to reach statistical significance. There was also neither a significant difference in the number of pHH3+/Dcx+ double-labeled cells, nor in the proportion of the pHH3+ cells that were also Dcx-positive (Fig. 5D,F). To analyze neuronal fate in the OB, the distributions of 2 GABAergic interneuron populations were quantified. Neither calbindin-positive (CB+) cells in the glomerular layer nor calretinin-positive (CR+) neurons in the glomerular and granule cell layers of the OB showed a significant difference in population size or distribution between genotypes (Fig. 5G,H).
To further quantify postnatal neurogenesis, BrdU labeling was carried out using a protocol that detects newborn neurons in the OB; after 6 consecutive days of BrdU administration from P7, the OBs of Visc-2−/− and Visc-2+/+ mice were analyzed at P28 (Supplementary Fig. 4A). The total number of BrdU-positive (BrdU+) cells in the granule cell layer was not significantly different between genotypes (Supplementary Fig. 4A–C). In addition, there was no variation in the number of BrdU+ cells that were also NeuN-positive by double-labeling, suggesting that there is no difference in the neuronal identity of these newly generated cells (Fig. 4B,D). Taken together, the combined OB data suggest that it is unlikely that Visc-2 disruption adversely affects the final destination or identity of newly generated neurons from the SVZ into the OB.
Finally, carbocyanine dye tracing experiments were carried out to examine axonal projections to the cortex at P7 from Visc-2−/− and Visc-2+/+ mice; specifically, from the cerebral peduncle to layer 5 (Fig. 6A–C) and thalamocortical projections from the ventrobasal complex (VB) to the primary somatosensory cortex (S1) (Fig. 6D,E). These data suggest that the same population of layer 5 projection neurons with similar proportions were backlabeled in Visc-2+/+ and Visc-2−/− brains (Fig. 6A). Both populations of these apical dendrites showed a comparable structural appearance and spine density as they reached the marginal zone with a terminal tuft (Fig. 6B,C). In addition, somatosensory cortex tracing demonstrated a similar pattern of thalamocortical projections in both Visc-2+/+ and Visc-2−/− mice (Fig. 6D). These projections crossed the internal capsule and striatum along comparable trajectories with a similar fasciculation pattern and formed the characteristic periphery-related pattern in layer 4 of the barrel cortex (Fig. 6E).
Although no overt pathological phenotype was evident, we continued to characterize Visc-2−/− mice using a battery of behavioral tasks on a cohort of age- and sex-matched wild-type and knockout animals. As a baseline assessment of activity as well as exploration in a novel environment, LMA testing was performed. No significant difference in the total distance traveled throughout the trial or during the initial habituation phase was observed between Visc-2+/+ or Visc-2−/− mice (Fig. 7A,B). There was also no difference in motor function as demonstrated by the performance on the accelerating rotarod (Fig. 7C), or in anxiety-related behavior as assessed in both the anxiolytic open field tests (Fig. 7D) and elevated plus maze (data not shown). With particular relevance to the expression of Visc-2 in the OB, the sense of smell was assessed using an olfactory preference task where both aversive and attractive scents are presented (Witt et al. 2009). Both wild-type and knockout mice were able to clearly distinguish between the 2 classes of odors, with similar amounts of time exploring aversive versus attractive scents in both genotypes (Fig. 7E). We went on to analyze cognitive performance using a number of tests of spatial memory. In the short-term exploratory spatial memory tasks—spontaneous alternation and Y-maze spatial novelty preference tests—both Visc-2+/+ and Visc-2−/− mice performed well above chance levels (Fig. 7F,G), with no significant difference in their activity in the apparatus (data not shown). In the Morris water maze test of spatial reference memory, there was no significant difference between genotypes in the rate of acquisition of the hidden platform position during 6 days of training based on swim path time (Fig. 7H). During the first probe trial on day 7, both Visc-2+/+ and Visc-2−/− mice spent significantly more time in the quadrant where the platform was originally placed, which indicates that both groups of mice had learned the location of the platform to a similar extent (Fig. 7I). In agreement with these data, a second probe trial after 3 further training days showed a slight improvement in the time spent in the target quadrant by mice of both genotypes, with no impairment in Visc-2−/− mice (Fig. 7I). Taken together, these data suggest that loss of Visc-2 has no overt effect on brain pathology or on behavior.
Sequences of noncoding Visc transcripts are not only conserved among mammals, but expression of Visc-2 orthologous transcripts occurs in the developing brain of marsupials, mice, and humans. Nevertheless, data from a range of neuroanatomical and behavioral analyses failed to identify physiological abnormalities in Visc-2−/− mice compared with wild-type controls. Despite a highly localized neurodevelopmental expression pattern in the SVZ, RMS, OB, and cortex, no defects in neuronal migration or anatomy were identified in Visc-2−/− animals, and no abnormal anxiety or cognitive behavioral phenotypes were observed.
One explanation for these data is that loss of Visc-2 is functionally compensated for by other transcripts in the region not influenced by the Visc-2 deletion strategy, such as the lncRNA Visc-3. Indeed, the transcriptional complexity of the noncoding RNA transcripts from this locus became clearer since the initiation of this study, with multiple lncRNAs as well as an mRNA all displaying specific neurodevelopmental expression profiles (Laneve et al. 2010; Shibata et al. 2011). It is noteworthy, therefore, that there was a small but significant reduction in miR-9-2 expression in the Visc-2−/− brain versus controls, suggesting that the deletion of the first exon of Visc-1 has a modest diminution of transcription of the pre-mRNA9-2, which is mostly transcribed from a separate, more proximal, promoter in a neural cell line (Laneve et al. 2010). This modest reduction in miR-9-2 expression is not expected to cause any phenotypic abnormalities, as none are reported in mice lacking this miRNA (Shibata et al. 2011). An alternative explanation for our findings is that the engineered genomic deletion of Visc-2 has inadvertently removed a regulatory element of miR-9-2, such as a specific chromatin modification; indeed, there is substantial evidence for active histone methylation (e.g., H3K4me3) in the brain across the majority of the Visc locus (Fig. 1C; Stamatoyannopoulos et al. 2012). Nevertheless, the Visc-2 deletion did not encompass the known Mef2c-binding sites upstream of the proximal pre-mRNA-9-2 promoter (Davila et al. 2014) nor did it significantly alter Mef2c expression levels (Fig. 4B).
That we were unable to identify a phenotype in Visc-2−/− mice can be explained in 2 further ways. First, it could reflect limitations in the screening assays that were unable to detect a bona fide phenotype. We emphasize that the choice of these assays was informed by the specific expression pattern of the Visc-2 lncRNA. Moreover, we applied a battery of behavioral, anatomical, and neuropathological tests that have been rarely, if ever, previously applied together to investigate a lncRNA knockout phenotype in the nervous system. However, it is not possible to rule out a subtle phenotype in Visc-2−/− animals that would only become apparent at a specific time point or within a defined neuroanatomical region or after environmental manipulations, either in the laboratory or in the wild, have been applied. Neuronal hyperexcitability and seizures, for example, were only manifested in Bc1 knockout mice upon auditory stimulation (Zhong et al. 2009). We note that there are many examples where highly evolutionarily conserved, developmentally expressed protein-coding genes have been knocked-out in the mouse, yet do not yield overt phenotypes despite considerable experimental scrutiny (Barbaric et al. 2007) and also examples of lncRNA knockout mice that fail to yield phenotypes (Nakagawa et al. 2011, 2012; Schorderet and Duboule 2011; Eissmann et al. 2012; Zhang et al. 2012; Sauvageau et al. 2013). It is also noteworthy that lncRNAs without evidence for evolutionary conservation can show phenotypes when disrupted. For example, the mouse-specific lncRNA Braveheart is important for cardiovascular lineage commitment (Klattenhoff et al. 2013).
Our results could also be explained if transcription of mouse Visc-2 has no biological consequence. The Visc-2 sequence is, however, highly conserved in human (67% identity) as well as opossum (66% based on local alignment), and its expression pattern is consistent in mouse, opossum, and human embryonic brain development which could reflect the conservation of its transcriptional regulation. Consequently, we believe it more likely that Visc-2 is functional but that the effect of its deletion upon fitness is insufficient to manifest itself in the assays screened. One possibility is that the Visc-2 DNA or RNA sequence acts in cis to subtly modulate the expression of nearby genes, such as Mef2c or pre-miR-9-2, or in trans to moderately alter the expression of more distal genes. Many point mutations in Visc-2 genomic DNA appear to have been deleterious; however, as its sequence has been preferentially conserved over mammalian evolution.
Many more lncRNA knockout studies combined with phenotypic analyses at the level of detailed scrutiny applied here will be required to establish the general significance of these transcripts in the context of a whole organism. For example, it has been demonstrated that deletion of 2 large noncoding regions of approximately 1 Mb containing over a thousand conserved noncoding elements, many of which will be transcribed, does not lead to detectable abnormalities in mice (Nobrega et al. 2004), although only gross physiological assessments were performed. Therefore, it is yet to be established whether there are categories of lncRNAs that are more likely to manifest in phenotypes when disrupted in mouse. Next-generation sequencing is a potential strategy for prioritizing noncoding candidates, and a number of lncRNAs have been identified with expression patterns corresponding to specific cortical layers (Belgard et al. 2011), or in the adult mouse SVZ neural stem cell lineage (Ramos et al. 2013). It will be interesting to determine whether these transcripts play important roles in neuronal fate determination in the developing brain; consequently, a high-throughput knockout program focussing on lncRNAs, similar to that currently underway for protein-coding genes, may be required (Bradley et al. 2012). One remaining difficulty is selecting the manner by which to disrupt lncRNA transcription in a mouse model without prejudicing the outcome of whether the transcript itself, the act of transcription, or even both, transact function (Hainer et al. 2011). To date, genomic deletions to remove regions of lncRNA exons and their putative promoters have been described elsewhere in mouse (Zhang et al. 2012). Yet, as discussed above, the loss of potentially confounding chromatin modifications must be considered. Potentially, such issues can be avoided by the insertion of transcriptional termination signals (Bond et al. 2009), and recent advances in zinc-finger nuclease and CRISPR/Cas9 technology may provide a more robust high-throughput approach (Gutschner et al. 2011; Mali et al. 2013). Illustrating the importance of these methodological issues, the lncRNA Fendrr has been disrupted using 2 independent strategies; although both methods resulted in a lethal phenotype, Fendrr deletion led to delayed development of the lungs (Sauvageau et al. 2013), while multisystem defects were observed after insertion of a transcriptional termination signal (Grote et al. 2013). Whatever strategies are employed, genetic disruption of lncRNAs in whole organisms will continue to be a vital tool to dissect their functional roles and their degree of influence on development and survival.
Supplementary Material
Supplementary material can be found at: http://www.cercor.oxfordjournals.org/.
This work was supported by the UK Medical Research Council, Biotechnology and Biological Sciences Research Council, UK and the European Research Council through an advanced grant (DARCGENs). Funding to pay the Open Access publication charges for this article was provided by the University of Oxford.
The authors thank David Bannerman and Amy Taylor for behavioral testing advice and assistance and Abigail Avelar and Anna Hoerder-Suabedissen for their neuropathological assistance. Conflict of Interest: None declared.


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Gallatin, TN
The Ruff-Life
The Ruff-Life
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T. Nichol T.
42 Reviews
16 Repeat Guests
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Overnight At Sitter's Home
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7 days notice required for bookings
Responds within 55 minutes
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Pet Requirements
1-20 lbs
20-40 lbs
40-80 lbs
80+ lbs
All Ages OK
Must Be Spayed or Neutered
Must Be Vaccinated
List of Breeds Not Accepted
Pit Bull - American Staffordshire Terrier, Pit Bull - Bull Terrier, Great Dane, Pit Bull - Staffordshire Bull Terrier, Pit Bull - American Pit Bull Terrier
T. Nichol's Resident Pets
Labrador Retriever
8 yr 1 mo old
Coton de Tulear
6 yr 1 mo old
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T. Nichol's House
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Medium Yard
100% fenced & locked
Smoke-free home
Pets allowed on furniture
Air-conditioned inside
Dog door access
About T. Nichol
10 Years Experience Caring For Dogs
Big or Small, we love 'em all!
We started using DogVacay a couple of years ago so that we could make sure, while we were on vacation, our dogs were well taken care of. Just like you, we decided to do a search for alternate boarding resources because, it just didn't sit well with us that our dogs would be confined to a kennel, in a potentially stressful environment, while we were out having
a good time. We knew that in order for us to thoroughly enjoy our vacation, we had to know our pets were just as comfortable as we were. DogVacay gave us that reassurance so, in return; we decided to open up our home.
Our home has a medium-sized, fenced in back yard that your dog(s) will have access to at all times. The exterior door leading from our laundry room has a built in pet door that allows dogs to enter and exit our home at their leisure. (If your dog is unfamiliar with using a doggie door, we will train them. Most dogs get the hang of it with in the first couple of minutes.) We promise a kennel free environment allowing your dogs to have free run of our home. They are welcome to sleep in our bed and will be loved as if they were our own. (We guarantee oodles of belly rubs and back scratches too!)
Weather permitting, we like to take the dogs for long hikes and even swimming at a nearby lake. We have a pup-mobile set aside specifically for all our dog outings. If you are comfortable with us transporting your dog(s) and allowing him to go "off campus", this is certainly something your dog can enjoy during his stay.
As a courtesy, pictures/videos will be sent to you daily so you can see just how comfortable your dog is while he is under our care.
Our dogs love meeting new friends. They grew up in a doggie day care, have never met a dog they didn't like and actually enjoy showing new dogs around our home. In no time at all, your dog and ours, will become the best of friends.
We are very hands on dog owners. We travel with our dogs, "talk" to our dogs and spend all our free time with them. We are extreme home bodies, so our dogs get a lot of human love and attention. We enjoy taking out the dog toys and playing with our dogs as most people would their kids. Your dog will receive the same love and attention here at The Ruff Life as we give our own.
We are constantly aware of the safety of the environment when walking our dogs, while they are in our home and while they are either inside (or out of) our fenced in yard. We make sure to see other animals, children or cars before our dogs do so that we can prevent potential accidents from occurring. We never leave food out and always separate dogs when feeding.
We keep our home clean and our pets clean. So, if your dog stays with us 5 nights or more, we promise to give him a complimentary doggy bath before your arrival. That's just one less thing you'll have to worry about when you get home :)
Thanks for taking the time to read our introduction. The concern you have for your dog and his comfort level is the same concern we have for ours. We promise to foster your dog(s) with the utmost care, love and responsible eye that we know you would. You can put your trust in us.
Thanks again...we look forward to hearing from you.
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T. Nichol T.
42 Reviews
16 Repeat Guests
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Overnight At Sitter's Home
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Boarding Overnight At T. Nichol's Home
Watches up to 1 dog /night
Dogs may be unsupervised for 2 - 4 hours/day
Dogs will have full access outside for potty breaks
1 walk/day
Pets can sleep wherever they want
Lauren E.
Aug 2016
We can't express enough how much we appreciate Nichol and Eric for always being willing to take care of our pup and loving on her! Highly recommend these sweet folks!!
Lauren E.
Aug 2016
Nichol and Eric are amazing as always with our pup, Hayley! They are such sweet people and Hayley always comes home tuckered out and happy as can be when she's stayed with them!
Tammy H.
Jun 2016
Myley has the best time when she goes on vacation with T. Nichol. I have been more than pleased with how well they take care of my baby. The pictures of her having fun playing are icing on the cake.
Susan T.
Jun 2016
Nichol and Eric are wonderful! My sweet large dog loved being with them and the other dogs. He was so happy when I picked him up. It was comforting
to know he was being cared for in a home environment and Nichol sent pics and videos to keep me updated and she was flexible when I needed to change drop off dates. I highly recommend them. Great experience....
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Pamela T.
Jun 2016
Guinness had a great time this weekend. He loves having easy access to the yard for romping. Nichol and Eric provide good supervision and seem to really love having their fur kid guests. Thanks.
Carol C.
May 2016
Nichol and her husband were awesome. My dog had never been to Dog Vacay before and he absolutely loved it and so did I. Nicole sent pictures to me on
a regular basis to let me know how my dog was doing and provided a warm, loving environment with loads of play time and interaction with other dogs. My dog came home so happy and worn out from playing. He will be going back on Memorial Day week for more good times. Love, love this home away from home for my Golden Retriever.
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Tammy H.
May 2016
Nichol and her family are great, both Myley and Gomie enjoyed their stay SO much. Definitely using them again.
Patti M.
Apr 2016
Wow, what a relief to find a dog sitter like Nichol and her family! While we were away, Nichol sent several photos and a video of our Bella that
really put our mind at ease. My husband and I felt very good about leaving Bella for the first time and Bella really seemed to enjoy herself especially with the opportunity to play with other dogs. Thank you, Nichol, for providing such a safe and loving stay for Bella. We really recommend Nichol; you can feel confident in leaving your dog with her and her family. Looking forward to the next doggie vacay! PS Thanks for the dog cookies and for giving Bella a bath. She smells so good and is nice and soft. I really have to get the name of that shampoo.
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Kris H.
Mar 2016
Amazing! Felt very comfortable leaving my little gal with this great family. Recd my pics that were reassuring that Ruthie was having a great time which allowed me to have an equally great time.
Barbara W.
Mar 2016
This was our first experience with Nichol and DogVacay. My dog Sam had a great time and we would highly recommend Nichol to anyone looking for a great experience for your pet when you can't take him with you
Marcie K.
Feb 2016
We found the perfect place for our Labrador retriever puppy! 5 months and very energetic.. Nicole invited another Lab to stay with Camo so they
could play, and play they did!! We were very grateful for the pics and videos!! I honestly couldn't tell them apart
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Dale N.
Feb 2016
Thank you for the wonderful stay! We wouldn't change a thing.
Tracy B.
Jan 2016
This was Laila's first overnight stay. Nichol sent pics showing how she was doing and was always quick to respond to my text messages. Laila will definitely be visiting again
Lauren E.
Jan 2016
Nichol and Eric were AMAZING with our dog, Hayley and were some of the sweetest people!... I was honestly afraid she would have wanted to stay there
forever! Nichol was so attentive in keeping our same schedule, needs, and training that we were currently doing and really treated Hayley like she was one of her own. I loved the fact that they were home a lot to keep company with the other fur-friends and they have a great backyard, so Hayley didn't miss us at all once we dropped her off. We (and Hayley!) can't wait for another opportunity to board with Nichol and Eric again!
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Andrew M.
Jan 2016
Nichol and Eric are just plain amazing. There is no other way to say it. We have been traveling a lot in the last few weeks (3 straight weekends) and
Jaeger has been taken care of so well! When we pull into their driveway he knows he is going to have an awesome time and he gets so excited. He loves them, and it is obvious that they love him too.
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Judy G.
Jan 2016
This is the most "pawsome" way to board my best friend, Tucker. I didn't worry about him at all, and I could tell from the pictures sent each day
that he was having a ball with his new-found friends. I just can't say enough about the care and love he received from Nichol!
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Valerie H.
Jan 2016
Nichol & Eric are very welcoming pet hosts! They both LOVE dogs, and you can tell by the way the dogs respond to them, the dogs in their are very
happy :-)
They go above and beyond to keep owners informed about their dog's status, sending texts and pictures each day.
At pick-up, Nichol gave us a bag of homemade dog biscuits for our little doggie to enjoy. She sure did!
We would definitely come back in the future. Thank you for making this a stress-free doggie vacay!
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Leah H.
Jan 2016
Let me start by saying I'm an overprotective fur mama, but I was so at ease leaving my pup here after the meet and greet at her home. I loved the
picture updates, the homemade dog biscuits, the environment, and I will definitely be using them again, and have been telling everyone about it! If you need a place for your four pawed loved one, look no further.
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Justin C.
Jan 2016
Fantastic! My dog, Minna, loved her!
Susan G.
Dec 2015
I was absolutely blown away by this experience! Nichol took in my Frankie Mae as if she were her own. I received updates , pictures, and video
showing just how much fun she was having. The dogs are inside and out, have the opportunity to play and socialize, and gets LOTS of love and TLC. This was a last minute need for us, as we were traveling for Christmas and our family fell through. Next time it will be a no brainer... Nichol is my new "go-to".
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Hannah M.
Dec 2015
Nichol was so great with Bucky! We will definitely be using them again!
Andrew M.
Dec 2015
Yet again Nichol and Eric provided an awesome stay for Jaeger.
Robyn M.
Dec 2015
Nichol is the BEST! We've left our two dogs with her two previous times, and she is amazing! This last visit while we were out of town, our older dog
was having some pain and discomfort--Nichol went out of her way to love on him and keep me updated with reports and pictures. We wouldn't trust our "babies" to stay with anyone else:)
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Pamela T.
Nov 2015
Nichol and Eric are wonderful hosts. My Guinness had a great time and has two new best friends in Wriggly and Mercy. Nichol and Eric were totally
fine with Guinness snuggling in the middle of the night. We received several pictures and it was clear our boy was having a good time. Nichol sent us home with yummy smelling homemade treats / I wanted to eat them myself. Thanks so much for making Guinness feel so at home.
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Regina .
Nov 2015
When I was looking for a place for my 16 year old German Shepard to stay while on vacation, I knew it would take someone special to care for her
because of her age. When I spoke to Nichol on the phone I could tell she had a really caring personality. When we went out to meet her I knew within a few minutes she was the kind of person I wanted to care for Woofie. She and Eric went above and beyond to make sure she was spoiled and pampered while we were away. She sent daily updates and pictures, which helped me relax and know she was in great hands. And did I mentioned she even spoon fed her at times to make sure she would eat due to her age. She is awesome and yes we would love to use her for any future trips we may take. This is a great service!!!!
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Andrew M.
Nov 2015
Nichol and Eric were fantastic. Jaeger really seemed to love his time with them, and loved the peace of mind that we had with him there. We will absolutely be using them again!
Pam I.
Oct 2015
LOVED, LOVED, LOVED leaving Gracie (our Jack Russell) with T. Nichol and DogVacay....I was a little nervous, but Nichol sent many photos of Gracie
during the day. When we picked Gracie up, Nichol had made out a card telling us about hers' and Gracie's experience. She even sent her home with homemade doggie biscuits, which Gracie loved! The experience was great...could not ask for nicer hosts. We have already booked Thanksgiving week for Gracie!
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Wendy P.
Oct 2015
My dog stayed with T. Nichol T for a week. We had a meet and greet before to see how our dog would do. We were very comfortable leaving Bella and
would recommend A Ruff Life to anyone. We received daily pictures and updates to keep us posted of Bella's activities. Thank you T. Nichol T!
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William A.
Sep 2015
Our only concern was that the dogs were so comfortable that they would not want to go home. From the photos we realized that they were in good hands
and that Nichol and her family were wonderful dog lovers. The best part was that we had no worries about the dogs while on VaKay in Nashville.
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Robyn M.
Sep 2015
Nichol is amazing and truly loves our pups! This is our second time having her watch our (2) dogs, and they LOVE staying at her house! She has a
great fenced in backyard where they are safe, but have lots of room to run around; and she sends daily updates and pictures to keep us informed about how they're doing. She's just THE BEST!!!
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Justin C.
Sep 2015
Nichol and Eric were fantastic! My dog, Minna, was truly on a vacation (and it allowed me to enjoy mine). I frequently received adorable pictures of
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They have a great backyard, and make their house very dog-friendly. They truly take care of your dog like a child.
At the end, I received the kindest note, along with some tasty treats for Minna (which I considered eating myself).
If you travel like me and happen to be going through Nashville, email them! You'll feel great knowing your pup is in good hands.
-Justin, California
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Tiffany A.
Sep 2015
Nichol and Eric watched Murphy for a week while we went on a trip and they were so great! We received pictures and videos daily and anytime I had a
question or asked about him, Nichol was quick to respond. The set up they have with the doggie door is perfect! Murphy caught on quickly and really enjoyed playing with their dogs. We wouldn't hesitate to use them again! Murphy didn't want to leave. Thanks for putting my mind at ease while we were away!
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Sep 2015
Our 14 year old Zoie was well taken care of by Nichol. We got pictures and updates everyday. Zoie didn't even act like she was ready to go home when I went to pick her up.
Brian H.
Aug 2015
This was the longest trip we've taken without our babies and honestly, it was so stress-free. She felt like family and we were so comfortable leaving
them with her. She truly loves dogs and it shows. Constant updates of them playing and having a great time! We would recommend her to anyone needing a friendly, caring environment and will definitely use her services again!
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scott k.
Aug 2015
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Tiffany A.
Aug 2015
Nichol and Eric were great to work with! Murphy had a blast going to their house for daycare. They stayed in contact with me throughout the day and
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Jul 2015
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Jul 2015
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Jul 2015
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National Day 2017 and 2018
National Day is the celebration of the independence and subsequent unification of the Emirates in 1971.
|2 Dec
|National Day
|3 Dec
|National Day Holiday
|2 Dec
|National Day
The United Arab Emirates (UAE) is a union of seven distinct Arab kingdoms located on the south eastern edge of the Arabian Peninsula and bordering the Persian Gulf. The history leading up to this event is complex. First, Arab pirates in the 17th, 18th, and 19th Centuries were accustomed to raiding British ships, which led the British to attack in 1809 and 1819. They then formed an alliance with local rulers beginning in 1820 and culminating in 1892. In exchange for British protection, certain limitations on independence were agreed to. Later, in the 1960s, the British withdrew, ending the longstanding "Exclusive Agreement" on December 1st, 1971.
No union was agreed upon in the days just before the end of the treaty, and Iran and Saudi Arabia began to act aggressively toward the tiny emirates. Out of necessity, Abu Dhabi and Dubai drew up a constitution and began a union immediately on December 1st. Four more emirates joined on December 2nd, and the last one joined in February of 1972.
Yawm Al Watani, translated "National Day," is celebrated every December 1st and 2nd to remember the events described just above. The country's first president, Sheikh Zayed bin Sultan Al Nahyan, who led the unification movement, is also honored at this time. Grand-sized National Day events occur all across the country every year, and most are off work for at least four consecutive days. The skies are lit up with profuse firework displays. Dancing and auto shows are common. "Heritage Villages" are erected all over the nation to remember traditional ways during this time. Concerts, parades, special museum exhibits, and more abound. The whole nation is thrown into a celebratory mood, and there are events to be found "around every corner".
National Day Events
Some of the most notable events to consider attending, though one cannot hope to list them all, are as follows:
- The Abu Dhabi Air Expo. The air show typically involves seven jets spewing out the colors of the UAE flag as they soar through the air high above the crowd. There are many stunts performed, and the people pack in tight along the seaside road to watch the action. It is well worth seeing.
- The Emirates Classic Car Festival. Held in Al Ain, this the Mid-East's premier showcasing of classic cars, defined as anything built before 1986. There are a wide variety of cars, trucks, and motorcycles to be seen, and no car-lover will be disappointed.
- Dubai National Day celebrations, held int he Mirdif City Center from November 30th till December 5th. There are folklore shows from all over the Gulf region, concerts with UAE stars, "cartoon character shows," and of course, endless fireworks.
- The Umm AL Quwain Car Parade, held on December 1st. Vehicles decorated in bright colors crowd the streets for an "impromptu parade" with profuse flag-waving and a barbecue to follow it up. You will hear much horn-honking and Emirati music playing loudly on car radios. Picnicking and fishing are the "quieter side" of this celebration.
Should you be in the UAE on National Day, you will have no trouble finding an event to attend. Knowing what the celebrating is all about and where to find the best events, however, will make the day far more enjoyable.
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Entry requirements
A good Honours Degree in a relevant creative subject, which may include Fine Art, Sculpture, Curating or equivalent professional/industrial experience with evidence of academic achievement. Applications must be accompanied by a research project proposal outlining their intended area of interest and proposed investigation.
Months of entry
Course content
The course encourages a broad based, experimental and curatorial approach to fashion design, reflecting an individual's particular interest. The course operates alongside the large full time undergraduate degrees in Fashion and Fashion Promotion and will be able to call upon facilities from other newly established fashion related MA programmes. Students on this course are encouraged to extend their projects beyond the accepted norms of conventional Fashion Design., and will be expected to develop their proposals, possibly in a collaborative way utilising both University and outside resources. Fashion Shaping at the University is taught by a well qualified and experienced team of academics and technical support staff.
Teaching involves a combination of lectures, seminars, tutorials, presentations and industry liaison, and assessment is continuous. Theory underpinning practice is developed simultaneously throughout each semester and Negotiated Agreements Parts I and II allow students to acquire the ability to develop and refine proposals, which underpin their major projects, and reflect on personal progress. Students are expected to contextualize their work within a current framework, and develop evaluative skills in order to articulate their point of view. Research Methods and Application introduces students to a range of analytical research tools, forming the basis for all in-depth self-directed projects on the programme. Exploration of contemporary issues directs a practical realisation in 2D and 3D outcomes, and an ability to consider various career opportunities in the creative industries, which may include exhibitions management and artist-designer.
Information for international students
See www.uclan.ac.uk/international
Qualification and course duration
Course contact details
- Name
- Course Enquiries
- firstname.lastname@example.com
- Phone
- +44 (0)1772 892400


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Professional Mentoring Report
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1 Introduction The process of assessment, whether formal or informal, intentional or unintentional, dominantly affects people, specifically students. In fact, the assessment ambiance that is conducted throughout a student's life is a vital element of guidance and learning…
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It is based on a sequence of questions, which could be used within an assessment relating to self assessment. 2 Self Assessment Currently I am enrolled for completing my bachelors in "Computing in Application and Support". However, my short term objective is to complete the current semester. Furthermore, my medium term objective is to complete my degree program successfully with terrific scoring points. Moreover, my long term objective is to become a computer teacher/ instructor. However, in the current scenario, I am in the process of completing my short term objective i.e. to complete the current semester with substantial scoring marks. Selecting my current field related to computing contains certain factor that inspired me to set my long term objectives as a computer teacher / instructor. 3 Personality Profile I have a great interest swimming. I like listening to all types of music and attend a variety of concerts and shows. I love to travel and experience different cultures and countries. I also have a flare in learning computing technology in which I am studying in the hope to take my interest as a career. In the year 2000, I complete my ECDL certification from Castleblayney College. In fact, I experienced interaction with computing devices via ECDL. ...
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Sourdough, San Francisco
Hello All,
Hello All,
This is my third sourdough bread-baking attempt. The first two loaves were baked as boules in a cast iron pot. The bread was delicious, and the crust was crunchy and chewy and had a bit of a shine to it. The scoring was a bit rough on the second loaf, but it was still a better loaf than I had expected.


text:
One of the most interesting elements of CyPhy's product design is that its UAVs (unmanned aerial vehicles) are tethered to a hand-held control system on the ground, rather than free-flying. The company's "microfilament" technology sends power to the UAV, meaning that its flight time is unlimited. It also transmits high-def video back down to the control system, and the company says that unlike wireless links, it cannot be jammed by an enemy.
Read the article here: Boston Globe


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Explore MDVIP Member Reviews
Feb 3, 2016
Dr. Head has been seeing me for over 10 years and I can say he's the most effective physician I've had over the last 45 years. I am a 30 year veteran of Diabetes and Dr. Head has encouraged me to strive for better control and treatment of the disease. He take a genuine interest in me both as a patient and a person. I enjoy my visits and appreciate his availability to any special needs I happen to have. He has a good understanding of all the medications I take and is always taking care to coordinate them with any other meds prescribed by other doctors. I hope to continue my relationship with Dr. head for several years to come.
Sep 22, 2015
I am a 66 year old female and due to my poor decision, hadnt had a physical for a few years. The MD/VIP physical discovered breast cancer and I am on the road to recovery! My husband and I joined MD/VIP to be able to continue with our family doctor of 30 years in Omaha, Dr. Gilbert Head, when his practice joined MD/VIP. We have faith in him, trust him and with my husbands diabetes and 2 spinal surgeries, wanted consistency with someone who knew us and was very familiar with our medical history. Dr. Head has been right there with us through my diagnosis and surgery, a very caring doctor! One of the features of this concierge program that I appreciate is being able to email my doctor with concerns that dont require an office visit. With diabetes in our family, the nutrition section of the website which plans menus and provides recipes is also a very valuable resource.
Sep 21, 2015
I have been under Dr Heads care for many years and I debated making the change when he changed his large workload and large number of patients and joined up with MDVIP. This allowed him and his staff the extra time and resources to give me a very thorough physical checkup. I credit this complete physical for finding a cancerous tumor in my bladder early and to get me on a treatment plan early. I feel the decision to stay with Dr Head was a Godsend.


text:
The How Artistic Are You? Test
Their result for The How Artistic Are You? Test ...
Advanced Artist
You scored 98% for Advanced. Great Job!
You have scored higher than 80% which tells me that you really know your art! All those art classes and trips to the museum must have sunk in more than you thought. I hope you had fun with this test, after all, isn't art supposed to be fun? I love feedback. I hope you will let me know if you liked or even didn't like my test!
Don't forget to rate my test! Thanks
Look for my other tests
I don't want to leave you wondering which ones you got right or wrong, so here is a list of answers. Compare them and see how you did!
1. The Mona Lisa
2. Pottery
3. Cubism
4. Paper folding
5. Blue pigment
6. Andy Warhol
7. Hue
8. The Scream
9. Two colors directly opposite one another on the color wheel
10. Marc Chagall
11. Fixative
12. Vincent Van Gogh
13. Loading a brush with 2 different colors
14. Henri Matisse
15. Futurists
16. Pottery
17. Pointillism
18. Starry Night
20. Abstract Expressionism
21. All of the above, No and Yes
22. Georgia O'Keeffe
23. Adhering paper, fabrics, string, etc . to a surface
24. All of the above, Pop, Advertising, Graphic art
25. Analogous
Their Analysis (Vertical line = Average)
They scored 98% on Advanced, higher than 87% of your peers.
They scored 96% on Amateur, higher than 82% of your peers.
All possible test results
Advanced Artist
You have scored higher than 80% which tells me that you really know your art! All those art classes and trips to the museum must have sunk in more than you thought. I hope you had fun with this te... Read more
Amateur Artist
You obviously like art but your score tells me you are still an "amateur" artist! Give yourself a pat on the back for trying, but you might want to go back and study up on your art history. I l... Read more
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Study, Double Blind, Randomized, Comparing Two Groups to Evaluate the Safety and Efficacy of Autologous Mesenchymal Stem Cells From Adipose Tissue (CMMAd) in the Treatment of the Faecal Incontinence
This trial is active, not recruiting.
|faecal incontinence
|infusion of mesenchymal stem cells from adipose tissue, infusion of placebo
|phase 1/phase 2
|Andalusian Initiative for Advanced Therapies - Fundación Pública Andaluza Progreso y Salud
|Iniciativa Andaluza en Terapias Avanzadas
|Start date
|October 2013
|End date
|December 2016
|Trial size
|16 participants
|Trial identifier
|NCT02292628, CMMAd/InFe/2011
This is a clinical trial phase I/II, double blind, randomized to compare in two groups of patients to evaluate the safety and efficacy of the autologous mesenchymal stem cells from the adipose tissue (CMMAd) in the treatment of the faecal incontinence.
|United States
|No locations recruiting
|Other countries
|No locations recruiting
hide locations and contact info
|Huelva, Spain
|H. Juan Ramón Jiménez
|no longer recruiting
|Madrid, Spain
|Fundación Jiménez Díaz
|no longer recruiting
|Málaga, Spain
|Hospital Regional U de Málaga
|no longer recruiting
|Sevilla, Spain
|Hospital U Virgen del Rocio
|no longer recruiting
Study Design
|Endpoint classification
|safety/efficacy study
|Intervention model
|parallel assignment
|double blind (subject, caregiver, investigator)
|Primary purpose
Primary Outcomes
Number of Serious Adverse Events
time frame: 12 months
Secondary Outcomes
Changes in the faecal incontinence diary
time frame: 12 months
Anorectal manometry
time frame: 12 months
Jorge-Wexner Score
time frame: 12 months
Faecal Incontinence Quality of Life
time frame: 12 months
Eligibility Criteria
Male or female participants from 18 years up to 80 years old.
Inclusion Criteria
- Males or females in the age group of 18-80 yrs.
- A unique internal sphincter defect and / or external (no more than 100 º), at any level of the anal canal, of any cause (except those described in the exclusion criteria).
- Severity of faecal incontinence of 12 or more in the Wexner Score and / or at least six episodes of faecal incontinence for a period of 28 days.
- Duration of faecal incontinence of at least two years prior to inclusion.
- Patients who are able to understand the requirements of the study, and willing to provide voluntary written informed consent.
Exclusion Criteria
- More of an external sphincter defect and / or at any level of internal anal canal.
- Have received treatment to faecal failure with bulking agents, neuromodulation or surgery.
- Current anorectal tumors.
- Current anal fissures.
- Anorectal stenosis
- Chronic pelvic or anorectal pain.
- Pregnant or 6 months postpartum.
- Medical history of infection with Human Immunodeficiency Virus (HIV) or any severe immunocompromised state or therapy immunosuppressive.
- Malignancies in remission for less than a year before the study. An exception to the basocellular carcinoma (BCC) in "remission" for less than a year.
- Bleeding diathesis or current anticoagulant therapy.
- Chemotherapy during the 6 months preceding the study.
- Previous radiation with evidence of radiation injury in the treated area.
- Participation in any other clinical study during the 3 months preceding the pre-study visit.
- Patients with other serious disorders.
- Patients with uncontrolled inflammatory disease or taking drugs prohibited in the protocol or active perianal disease.
Additional Information
|Official title
|Study Phase I/II, Double Blind, Randomized, Comparing Two Groups to Evaluate the Safety and Efficacy of Autologous Mesenchymal Stem Cells From Adipose Tissue (CMMAd) in the Treatment of the Faecal Incontinence
|Principal investigator
|Fernando de la Portilla, Dr.
|In the phase of recruitment, of 20 months of duration, there will be included 16 participants who will be randomized to one of the groups of intervention (8 patients group CMMAd / 8 patients group placebo). The phase of follow-up of every patient will be 12 months, though, provided that these patients are seen regularly in the study sites of the study, once concluded his follow-up will be continued to be able to obtain safety information and efficacy to 24 months.
Trial information was received from ClinicalTrials.gov and was last updated in December 2015.
Information provided to ClinicalTrials.gov by Andalusian Initiative for Advanced Therapies - Fundación Pública Andaluza Progreso y Salud.
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Optoelectronic devices
Found: 16 products← Back to category "Optoelectronic devices"
Optoelectronics domestic
30D101B 3LS339A 5P142B AL307ZHM AOT102A KIPD35V-L 30T110A 3LS340A 5P142V AL307KM AOT102B KIPD40V-K 30T11 Read more
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Delivery fromRussia
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Display phones
Site: rt-komplekt.ru
Multifunction optical reflektomer of FOD-7202
The field, protected, pocket, easy optical reflektomer of FOD-7202. Odnomodovyy1550nm. Distances to 70 km and more. Displays the reflecting and nonref Read more
Verified partner for over 8 years
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Delivery fromUkraine
Display phones
Site: svpribor.com.ua
Thermoelectric coolers of a series MC
Thermoelectric coolers of a series MC are developed for cooling of tiny electronic components of different function. Read more
Delivery fromUkraine
Optoelectronic AOM3 devices
Optoelectronic AOM3 devices - these are the devices using the electromagnetic radiation of optical range in work. They accept, will transform and g Read more
Delivery fromUkraine
Gloss sensors
Sensors distinguish brilliant and opaque surfaces. Examples of application Recognition of transparent labels on The White Ribbon, glass and a plastic Read more
Delivery fromRussia
Optotiristora TO132, TO142.
Recommendations at installation: from damages to make the soldering of the isolated conclusions for protection of an optotiristor during time no Read more
Delivery fromRussia
Delivery fromJapan
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Trading platform Allbiz offers to see the catalog Optoelectronic devices which contains 16 products offers from 2 companies and enterprises. Don't know what Optoelectronic devices to buy? You can clarify the specifications, see photos Optoelectronic devices and choose the best seller and supplier. Buy Optoelectronic devices trough online ! On Allbiz — You can just place your order right on the site.


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Demand for shoes punched for City head nails brought forth the Kerckhaert SX Series. The SX-8 unclipped steel shoe is a 5/16" thick shoe with graduated widths. These shoes are used for trail, jumping, dressage and ranch work. Shoes are symmetrical. Sole relief on fronts.
Punched for 5 City, 5 Slim or 5 Combo. Sizes 0, 00, 000, 1, and 2 available; front and hind. Sold per pair.
QC Part #: 441735
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