A systematic and comprehensive phenotyping platform has been developed by the RIKEN ENU-mutagenesis project between 1999 and 2007. As a result of phenotype screening on this platform, we have discovered about 400 mutants as animal models for human diseases. All information regarding these mouse mutants is now available to the public through our home page (http://www.brc.riken.jp/lab/gsc/mouse/indexJ.html). In 2008, we reconstructed the existing phenotyping platform and built a new platform. The new system has a hierarchical structure, consisting of a fundamental pipeline that utilizes the existing platform and an additional pipeline, which is optimized for more in-depth phenotyping assays. Using this system, we have started to perform more comprehensive phenotyping of mouse mutants. We have opened this system to Japanese scientists as the Japanese Mouse Clinic. It is anticipated that existing mouse mutants will be reevaluated as disease models by identifying novel phenotypes on the new platform. We will share detailed information about the standard operating procedures (SOPs) of our phenotyping analyses with other related large-scale projects, such as the European Mouse Disease Clinic (EUMODIC) and the German Mouse Clinic (GMC). Moreover, we will contribute to international efforts to standardize mouse phenotype data by sharing annotation of mutant phenotypes, which are made by internationally standardized methods, with other related projects.

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The Japanese macaque (Macaca fuscata), along with rhesus and long-tailed macaques, is one of the macaca species. In Japan, it has been preferred for use as a laboratory animal, particularly in the field of neuroscience, because of its high level of intelligence and its gentle nature. In addition, the species has a relatively homogeneous genetic background and field researchers have accumulated abundant information on the social behavior of wild Japanese macaques. As future neuroscience research will undoubtedly be more focused on the higher cognitive functions of the brain, including social behavior among multiple individuals, the Japanese macaque can be expected to become even more valuable as a laboratory animal in the near future. The Ministry of Education, Culture, Sports, Science and Technology has launched a National BioResource Project (NBRP) to establish a stable breeding and supply system for Japanese macaques for laboratory use. The project is in progress and should lead to the establishment of a National Primate Center in Japan, which will support the supply of monkeys as well as social outreach and handling of animal welfare issues.

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The study of marine invertebrates is useful in various biological research fields. However, genetic analyses of these animals are limited, mainly due to difficulties in culturing them, and the genetic resources of marine invertebrates have not been organized. Recently, advances have been made in the study of two deuterostomes, an ascidian Ciona intestinalis and a feather star Oxycomanthus japonicus. The draft genome sequence of Ciona intestinalis has been determined, and its compact genome, which has less redundancy of genes compared with vertebrates, provides us with a useful experimental system for analyzing the functions of genes during development. The life cycle of Ciona intestinalis is approximately 2-3 months, and the genetic techniques including a perfect inland culture system, germline transformation with a transposon Minos, enhancer detection and insertional mutagenesis, have been established. The feather star Oxycomanthus japonicus conserves the characteristics of the basic echinoderm body plan with a segmented mesoderm, which is a fascinating characteristic for understanding the evolution of echinoderms. Oxycomanthus japonicus shows strong regeneration ability and is a suitable subject for analysis of the mechanisms of regeneration. In consideration of these features, the National BioResource Project (NBRP) has started to support the supply of wild-types, transgenic lines and inbred lines of Ciona intestinalis and Oxycomanthus japonicus.

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Hatano high- and low-avoidance (HAA and LAA) rats are separated by breeding from Sprague-Dawley rats by high versus low rates of avoidance responses in a shuttle-box task. In addition, compared to HAA rats, LAA rats show lower running-wheel activity, later sexual maturation, 5-day estrous cycling, lower sperm motility, more pronounced immunological reactions, and are generally less reactive to stress. The present study was designed to compare the effects of transmaternal exposure to genistein on these characteristics between HAA and LAA rats. To this aim, litters from both strains were fostered onto Sprague-Dawley rats receiving genistein by gavage with 5 mg/animal/day from day 17 of pregnancy through day 21 of lactation. Inhibited growth after weaning and reduced uterine weight at weaning were observed in the LAA offspring reared by genistein-treated dams. IgM antibody production in response to sheep red blood cells was significantly decreased in the HAA offspring reared by genistein-treated dams. During restraint stress, the plasma concentration of corticosterone was significantly lower in the LAA offspring reared by genistein-treated dams. Strain-related differences were detected in shuttle-box avoidance performance, running-wheel activity, estrous cycling, and sperm motility. The results demonstrate that transmaternal exposure to genistein potentially affects the immunological and stress responses as well as the post-weaning growth of the offspring. It suggests that a comparative study using Hatano rats would be useful for studying the influence of endocrine active chemicals on the whole body systems.

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Retinopathy and choroidal angiopathy were both detected in aged male rats of the WBN/Kob strain with sustained diabetes. Hyperglycemia and glucosuria were found starting from 12 months of age and lasted through 24 months of age. Macroscopically, the vitreous body was partially or entirely replaced by white mass in 3 of 9 diabetic males. Histopathologically, the intravitreal white mass consisted of collagen fibers accompanied by numbers of newly formed vessels. Intraretinal angiopathy was accompanied with newly formed vessels, which were observed within the retina in 5 of 9 diabetic males, and marked hyalinization of intraretinal vessels was detected in 6 of 9 males irrespective of the presence of intravitreal neovascularization. Furthermore, hyperglycemia-related choroidal angiopathy was also seen with newly formed blood vessels originating from the choroid penetrating the retinal pigment epithelial layer and invading the retina in 8 of 9 diabetic males. Focal proliferation or degeneration of the pigment epithelial cells was associated in the region with choroidal angiopathy. In females, choroidal vessels slightly raised the pigment epithelial layer; however, they were localized in the choroid. The present study indicates that the WBN/Kob strain of rats is a useful model for both diabetic retinopathy and diabetic choroidal angiopathy.

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The effects of intracerebroventricular (i.c.v.) administration of pituitary adenylate cyclase activating polypeptide38 (PACAP38) on prolactin (PRL) secretion and the activity of tyrosine hydroxylase (TH) were examined in adult male and lactating rats with or without suckling stimulus. In adult male rats and lactating rats with suckling stimulus, administration of PACAP38 (0.25 or 1 nmol) decreased PRL secretion and increased the activity of TH in the stalk-median eminence. On the other hand, the injection of PACAP38 did not affect PRL secretion and TH activity in lactating rats without sucking stimulus. Administration of PACAP6-38 (4 nmol), a specific receptor antagonist, also had no effect on PRL secretion and TH activity in adult male rats. These results suggest that i.c.v. administration of PACAP inhibits PRL secretion mediated by dopamine neuron within the hypothalamus, but the effects of PACAP differ depending on the physiological condition of animals. These observed effects of PACAP on PRL release may be pharmacological responses rather than physiological responses.

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We investigated the prevalence of Helicobacter hepaticus, murine norovirus (MNV), and Pneumocystis carinii and the efficacy of cross-fostering for their eradication in 49 genetically engineered mouse (GEM) strains at our institute. Prevalences of H. hepaticus, MNV, and P. carinii were 33.9, 36.5, and 8.6%, respectively, and immunodeficient strains showed relatively higher prevalence of the 3 pathogens than immunocompetent strains. Additionally, the same immune phenotype strains showed similar prevalences. Furthermore, it was found that NKT cells might play a role in H. hepaticus resistance. Interestingly, there was a high incidence of H. hepaticus and MNV multiple infection. Strains with single or multiple infections of H. hepaticus, MNV, and/or P. carinii were selected, and cross-fostering was conducted. Cross-fosterings were successful at eradicating P. carinii, but there were some failures for H. hepaticus and MNV, and the efficacy of eradication was relatively low compared with previous studies. We thought that this low efficacy might have been due to persistent infection and the high suscepibility to H. hepaticus and MNV of immunodeficient GEM strains. Therefore, cross-fostering may be appropriate for P. carinii eradication, but be inappropriate for repopulation of a new breeding colony with H. hepaticus or MNV infected GEM strains. Our findings provide basic data on maintenance, strain susceptibility, and successful rederivation, especially for GEMs.

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This study was planned to investigate the neuroprotective potentials of three commercially available prostaglandin analogues (PGA), in the ischemia and reperfusion model (I/R). Thirty New Zealand rabbits were divided into 5 groups and except for the control group (non-ischemic, non-treated), 0.9% NaCl, bimatoprost, latanoprost, or travoprost were applied to both eyes of animals of the respective groups for 1 week. At the end of treatment, ischemia was induced in both eyes of the 4 treatment groups by anterior chamber irrigation of the animals for 60 min. Following 24 h reperfusion, the animals were sacrified. Enucleated eyes and retinal tissues were investigated by light microscopy, electron microscopy, immunohistochemicstry for retinal histopathology, intracellular and apoptotic cells and by retinal morphometry. Vitreous samples were biochemically investigated for probable role of reactive oxygen species, by measuring xanthine oxidase (XO) activity. Analysis of morphometric measurements and vitreous XO activity revealed significant differences between the PGA-treated groups and the NaCl-treated group (P<0.05). Similarly, apoptotic cell counts in different retinal layers showed that PGA-treated groups had fewer apoptotic cells in all retinal layers than the NaCl-treated ischemic group (P<0.05). PGA may have high protective potential for different retinal layers and cells. Biochemical analysis of vitreous showed that all PGAs decreased vitreous XO activity significantly compared to the NaCl-treated group (P<0.05). However we could not find any statistically significant differences among the analogues. PGAs may reduce the injury induced by I/R, through the inhibition of XO activity, and it seems that their effects are elicited through numerous pathways.

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Serine palmitoyltransferase (SPT) is the enzyme which catalyzes the first step of the biosynthesis of sphingolipids. However, the precise roles of SPT in vivo are not well understood, since complete knockout (KO) of genes which compose SPT results in a fetal lethal phenotype. A conditional KO (cKO) mouse of SPT long chain base 2 (Sptlc2) was therefore developed, and the effects of Sptlc2 deficiency were examined. Single cell necrosis in the epithelia of the crypts of the small and large intestines was observed as early as 24 h after induction of knockout. At 48 h after induction, decreases in spleen and thymus weights and decreases in numbers of reticulocytes and lymphocytes were observed in cKO mice, and single cell necrosis in the intestine became prominent. At 72 h after induction, decreases in body weight, spleen and thymus weights, and numbers of reticulocytes and lymphocytes became obvious in cKO mice. Histologically, atrophy of gastrointestinal mucosa and lymphoid necrosis as well as depletion of lymphoid and hematopoietic tissues were observed. These findings suggest that SPT plays important roles in the maintenance of the gastrointestinal mucosa, especially in the proliferation of the mucosal epithelial cells, and that deficiency of Sptlc2 induces necrotic lesions in gastrointestinal cells followed by atrophic change of the tissue in short term.

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repro23 is an autosomal recessive mutation of the mouse generated by the N-ethyl-N-nitrosourea (ENU)-induced mutagenesis program at The Jackson Laboratory. The repro23/repro23 homozygous mouse shows male-specific infertility caused by defective spermatogenesis. In the present study, we investigated the testicular pathology of the affected mouse and performed linkage analysis to determine the chromosomal localization of the repro23 locus. Histological examination of the affected testis showed that the seminiferous epithelium of the repro23/repro23 mice contained spermatogonia and early stage spermatocytes, but no spermatids or spermatozoa. Immunohistochemical staining for Hsc70t, a spermatid specific protein, confirmed the absence of elongating spermatids. These findings indicated interruption of the spermatogenesis during meiosis in the repro23/repro23 mouse. By linkage analysis using 137 affected mice of F₂ progeny obtained from crosses between repro23/repro23 female and JF1/Ms (+/+) male mice, the repro23 locus was mapped to 2.2-Mb region of mouse chromosome 7. Although this region contains several potential candidate genes for the repro23 mutation, no gene already identified as a cause of defective speramatogenesis was in this region. Therefore, the gene responsible for the repro23 mutation is suggested to be a novel gene which plays an essential role in mammalian spermatogenesis.

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Silicone tubes are commonly used as a tool for long-term steroid treatment in animals. However, the release rates of steroids from these tubes have not yet been accurately determined. We measured the amounts of 4-nonylphenol (NP) remaining in tubes implanted in mice to obtain accurate release rates for original amounts of 20 or 1,000 μg NP in silicone tube implants. We achieved an accurate figure for the release rate from the tube, calculated using the total recovery from both the contents of the tube and the silicone matrix of the tube. The regression curve for the remaining amount of NP, as a percentage of the original amount (1,000 μg) was calculated as y (%) = 83.033 - 3.4722t + 0.0375t², where t is the number of days after implantation. The mean release rate over 56 days was 1.6% of the original amount per day.

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Although Tritrichomonas muris is a common parasite often detected in experimental animals including mice, its pathogenesis in host animals remains unclear. Proteomics can be used to specifically analyze biochemical host-parasite interaction and immune responses of the host to parasites. However, proteomics have not yet been applied to T. muris studies. Here, the effects of T. muris on the host were analyzed by proteomics. We found that 10 different proteins were expressed in T. muris-infected mice intestines compared with non-infected intestines. The identified proteins represented several functions mainly related to stress, immune response, metabolism and signal transduction. The results suggest that T. muris infection may affect processes that are acclimatizing to the environmental changes caused by the infection in the mouse intestine.

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To investigate the physiological roles of nerve growth factor (NGF) in the oviduct of golden hamsters during the estrous cycle, the localization of NGF and its receptors, trkA and p75, were determined by immunohistochemistry. Positive staining of NGF, trkA, and p75 was present in epithelial cells and muscle cells of the infundibulum, ampulla, and isthmus in the oviduct. The intensities of the immunohistochemical signals for NGF, trkA, and p75 did not markedly change in any segment of the oviduct during the estrous cycle. These results suggest that NGF may play autocrine/paracrine roles in oviductal transport, fertilization, capacitation of spermatozoa and early embryonic development in the oviduct of golden hamsters.

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Preimplantation development is critical for successful implantation and pregnancy. In the mouse preimplantation embryo, the first event of morphological and cellular differentiation is established during polarization and compaction at the 8-cell stage. The considerable cell surface and cytoplasmic changes and formation of different populations of cells at the 8-cell stage are fundamentally important for the development of all organisms. To determine genes that are specifically expressed at this crucial stage of embryo development and also to shed light on the different mechanisms that could be of importance during embryo development, we investigated mouse 8-cell and 4-cell embryo stage-specific genes using Digital Differential Display (DDD). The 8-cell stage-specific genes were sorted according to their ontology data from the Database for Annotation, Visualization and Integrated Discovery (DAVID), which outlines possible roles for the genes expressed at the 8-cell stage. This study highlights how online tools can be used to identify genes involved in embryo development. Identification of the 8-cell embryo stage-specific genes would open new opportunities for understanding molecular networks during the mid-preimplantation gene activation. Using bioinformatic tools, such as Digital Differential Display and DAVID, it will be possible to identify genes expressed at the 8-cell stage that are likely to be involved in mammalian preimplantation embryo development. Our results may provide a new foundation for molecular control at the onset of embryonic development in mammals, and should be of interest to the scientific community.

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The purpose of this study was to determine the usefulness of large rabbits for basic vascular interventional radiology (IR) experiments. We used 5 Akita large rabbits (Akita) and 5 Japanese white rabbits (JW). We conducted measurements of vessel diameters such as the aorta, and the iliac, renal, superior mesenteric, celiac, and proper hepatic arteries, and of the growth rates of VX2 liver tumors. There were significant differences between Akita and JW in the diameters of the thoracic aorta, lower abdominal aorta, and celiac artery. In other blood vessels, no significant differences were found. There was no difference in the growth rates of the VX2 tumors between Akita and JW. The possibility that Akita large rabbits could be utilized for vascular IR was demonstrated.

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