The Cell Engineering Division of RIKEN BioResource Center is a not-for-profit public "cell bank" that accepts donations and deposits of human and animal cell materials developed by the life science research community. We examine, standardize, amplify, preserve, and provide cell materials to scientists around the world. The major cell materials used around the world have been cultured cell lines, i.e., immortalized cells. Most human cell lines are derived from tumor cells. There is no doubt that the demand for these cell lines will never cease in the field of biology. In addition, stem cell lines such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are of great value in current biology and medical science. Thus, we are extensively collecting such stem cell lines, aiming at contributing to the fields of developmental biology and transplantation/regenerative medicine. In addition, the demand for primary cells has recently increased. To meet this demand, we have started the banking of primary human cells including somatic stem cells, such as umbilical cord blood cells and cultured mesenchymal cells. The staff of the Cell Engineering Division conduct not only the banking of cell materials, but also research and development relating to cell materials, such as the establishment of novel human and animal-derived cell lines and the development of new technology to utilize cell materials.

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The zebrafish is the simplest model vertebrate amenable to genetics, and genome information and methods of embryo manipulation have been accumulated worldwide. The numbers of mutant and transgenic zebrafish strains are rapidly increasing, and these strains will play important roles in the basic biology research and as model systems of the human diseases in the future. Although researchers who had established zebrafish strains, were distributing the fish on a discretionary basis, a well-established system for distributing the strains did not exit in Japan prior to 2003. Due to these circumstances, a system to collect, preserve, and provide zebrafish strains was established as part of the National BioResource Project useful model vertebrates in Japan and to the world.

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Medaka (Oryzias latipes) is a small freshwater teleost fish that serves as a model vertebrate organism in various fields of biology including development, genetics, toxicology and evolution. The recent completion of the medaka genome sequencing project has promoted the use of medaka as a comparative and complementary material for research on other vertebrates such as zebrafish, sticklebacks, mice, and humans. The Japanese government has supported the development of Medaka Bioresources since 2002. The second term of the Medaka Bioresource Project started in 2007. The National Institute for Basic Biology and Niigata University were selected as the core organizations for this project. More than 400 strains including more than 300 spontaneous and induced mutants, 8 inbred lines, 21 transgenic lines, 20 medaka-related species and 66 wild stock lines of medaka are now being provided to the scientific community and educational non-profit organizations. In addition to these live fish, NBRP Medaka is also able to provide cDNA/EST clones such as full-length cDNA and BAC/fosmid clones covering 90% of the medaka genome. All these resources can be found on the NBRP Medaka website (http://shigen.lab.nig.ac.jp/medaka/), and users can order any resource using the shopping cart system. We believe these resources will facilitate the further use of medaka and help to promote new findings for this vertebrate species.

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Regular monitoring of commercial laboratory rodents and institutional research animal residents is essential for microbiological quality control programs. The objective of our study was to investigate the recent prevalence of infectious pathogens in laboratory mice from eight experimental animal vendors and 56 institutional animal facilities in Korea. Our investigation was conducted in 2006-2007. Specific Pathogen Free (SPF) mice from four commercial breeders were clean according to serological, bacteriological, parasitological, and histopathological examination results. However, mice from one intermediate vendor that distributed SPF animals from main commercial vendors to local districts had Syphacia obvelata and Mycoptes musculinus infections. Additionally, mice from conventional animal breeders were highly contaminated. Among the 56 institutional animal facilities, mouse hepatitis virus (MHV), Sendai virus and Mycoplasma pulmonis positive results were obtained in 23.2, 8.9, and 1.8% of animals tested, respectively. These results indicate that quarantine and eradication efforts of infectious pathogens in these facilities are sub-optimal and need to be improved. The use of commercial conventional mice for research should be eliminated and appropriate vendor selection as well as thorough quarantine before releasing animals into a facility are needed. Finally we suggest qualified veterinary experts are needed at each animal facility to ensure an adequate health surveillance program.

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WMN/Nrs inbred rats have been widely used in radiation biology for years. However, their reproductive profile has never been examined. We examined various reproductive characteristics of WMN/Nrs inbred rats such as superovulatory response, oocytes spontaneous activation (OSA), and embryo development in vitro and in vivo. Superovulation was induced in 3- to 9-week-old females by injection of 150 IU/kg PMSG and 150 IU/Kg hCG by 48 h apart. Only 8- and 9-week-old animals superovulated averaging 31.4 and 43.9 oocytes, respectively, and superovulation did not depend on estrous cycle. Animals 3-7 weeks of age did not superovulate. Because Wistar strains have been known to show a high incidence of OSA, factors expected to affect OSA in WMN/Nrs, including the time interval of various steps from euthanasia to oocyte recovery, incubation media, estrous cycle, and anesthetic treatments, were examined. The time from animal euthanasia to oviduct excision was the only factor shown to affect OSA. We also compared in vitro and in vivo embryo developmental competence between embryos obtained by natural ovulation and superovulation. Although percent in vitro development of 2-cell embryos to blastocysts was similar for embryos obtained by natural ovulation (63.7%) and superovulation (69.7%), fetus development after oviductal transfer of 2-cell embryos was significantly lower in embryos obtained by superovulation than in those obtained by natural ovulation (60.2% vs. 87.5%, P=0.02). Our results provide important normative data regarding future applications of rat assisted reproductive technologies (ARTs) such as in vitro fertilization and cryopreservation in WMN/Nrs strain and may be applicable to other strains of laboratory rats.

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To evaluate murine norovirus (MNV) infection in laboratory mice, we attempted to develop an enzyme-linked immunosorbent assay (ELISA) system and an indirect immunofluorescent antibody (IFA) assay for detecting the anti-MNV-S7 antibody in mice. MNV-S7, which was isolated in Japan, was used in both assays. The antigen for ELISA was prepared by ultracentrifugation of culture supernatants of RAW 264 cells infected with MNV-S7. Positive sera were obtained from 6-week-old, female C57BL/6JJcl mice inoculated orally with MNV-S7. IFA against infected RAW 264 cells was able to discriminate positive sera from negative sera. Indirect ELISA was performed using 96-well ELISA plates coated with formalin-treated MNV-S7 antigen. In this ELISA system, mouse sera obtained 2 weeks after infection or later showed significantly high OD values and were judged positive. An equal level of anti-MNV-S7 antibody response was observed in BALB/cAJcl, C57BL/6JJcl, DBA/2JJcl, and Jcl:ICR mice; whereas, C3H/HeJJcl mice demonstrated slightly lower antibody production 4 weeks after infection. We also used this ELISA system to evaluate 77 murine serum samples obtained from 15 conventional mouse rooms in research facilities in Japan and found that approximately half of the serum samples contained antibody to MNV-S7. We found that some serum samples were negative for antibodies to mouse hepatitis virus and Mycoplasma pulmonis but positive for antibody to MNV-S7. The results suggest that the MNV infection is more prevalent than other infections such as mouse hepatitis virus and Mycoplasma pulmonis in conventional mouse colonies in Japan, as is the case in other areas of the world.

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In humans, hearing is a major factor in quality of life. Mouse models are important tools for the discovery of genes responsible for genetic hearing loss, often enabling analysis of the processes that regulate the onset of deafness in humans. Thus far, at least 400 deafness mutants have been discovered in laboratory mouse populations and used in the study of deafness. Here we report the discovery of a new spontaneous recessive Rinshoken shaker/waltzer (rsv) mutant derived from our in-house C57BL/6J stock, which exhibits circling and/or head-tossing behaviour and complete lack of auditory brain response to any sound pressure. The hearing and balance phenotypes are associated with structural defects, in particular, disorganisation and fusion of stereocilia in the inner ear hair cells. Two sets of intersubspecific N₂ mice were generated for the positional cloning of the rsv mutation. The mutant locus was mapped to a 4.8-Mb region of chromosome 9, which contains myosin VI (Myo6), a gene responsible for deafness in humans and Snell's waltzer mutation in mice. The rsv mutant showed reduced expressions of Myo6 mRNA and MYO6 protein in the inner ear. Moreover, no immunoreactivity was observed in the cochlear and vestibular hair cells in the rsv mutant mice. We sequenced the genomic region (30,154 bp) of Myo6, including all coding exons, a non-coding exon, UTRs and the Myo6 promoter; however, no mutation was discovered in these regions. We therefore speculate that loss of MYO6 expression might cause shaker/waltzer behaviour and deafness in the rsv mutant; also, loss of MYO6 expression might be the result of mutations in an unidentified regulatory region(s) of the gene.

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Obesity, hyperglycemia, hyperlipidemia, and diabetes-associated complications appear at younger ages (6-8 weeks) in the male Spontaneously Diabetic Torii-Lepr^fa (SDT-fa/fa) rat than in the male original SDT (SDT-+/+) rat. However, the incidence and progression of diabetes mellitus and diabetic complications in the female SDT-fa/fa rat have not been reported in detail. In the present study, the pathophysiological features of the female SDT-fa/fa rat were examined, and compared with those of the female SDT-+/+ rat. Female SDT-fa/fa rats showed hyperphagia, obesity, hyperglycemia, and hyperlipidemia from 5 or 6 weeks of age, and hyperinsulinemia was observed from 5 to 12 weeks. Pathological changes pancreatic islets were observed from 8 weeks. Renal function parameters, such as urine volume and urinary protein, increased from 16 weeks, and pathological findings in the renal tubule, and cataracts were also observed from 16 weeks. Increases of visceral and subcutaneous fats were obvious during the observation period. In pair-feeding with SDT-+/+ rats, SDT-fa/fa rats showed improved hyperglycemia and hypertriglycemia, but hypercholesterolemia was not entirely improved during the study period. Female SDT-fa/fa rats showed diabetes mellitus and diabetes-associated complications at young ages, and fat accumulation was remarkable. Suppression of hyperphagia in SDT-fa/fa rats was effective at improving hyperglycemia and hypertriglycemia. In conclusion, the female SDT-fa/fa rat has the potential to become an important animal model of type 2 diabetes mellitus with obesity, especially for women, for which few models currently exist.

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The present study was designed to evaluate the time-dependent effects of Nanpao, a kampo medicine, on age-related changes in the estrous cycle of female rats, and to investigate the utility of measuring electrical impedance in the vagina (EIV) for studying transitional changes in the estrous cycle. Rats were allocated to 3 groups: control, Nanpao 30 mg/kg/day, and 100 mg/kg/day groups. EIV measurements and cytology samples were taken for 14 days at the age of 6 months before the initial treatment. After the start of the treatment, these data were collected at about monthly intervals until the age of 10 months in the same manner. Observations at the ages of 7 (weeks 2-3 of dosing) and 8 months (weeks 6-7 of dosing) showed that loss of a regular estrous cycle in the 100 mg/kg/day group was inhibited as compared to the control group. Moreover, at the ages of 9 (weeks 11-12 of dosing) and 10 months (weeks 17-18 of dosing), these effects were identified not only in the 100 mg/kg/day group, but also in the 30 mg/kg/day group. Since vaginal cytology and EIV gave almost concordant results as indicators of estrous cyclicity, we concluded that the measurement of EIV was capable of detecting time-dependent changes in the estrous cycle as well as observations of vaginal smears. A short period of Nanpao administration inhibited loss of regular estrous cycles, and the EIV method is a worthwhile approach to a more precise study of estrous cyclicity in rats exhibiting abnormal estrous cycles.

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The rdw rat is a hereditary hypothyroid strain isolated from Wistar-Imamichi rats. In the present study, adrenocorticotropic hormone (ACTH) and corticosterone responses to restraint stress (120 min) were examined in rdw adult male rats. ACTH response to restraint stress was higher in rdw rats than in hetero control rats. The plasma concentrations of corticosterone were lower in rdw rats than in control rats during the first 30 min after the onset of stress. Both ACTH and corticosterone responses to restraint stress in rdw rats recovered to control levels after thyroxine (T4) replacement therapy. These results suggest that hereditary hypothyroidism causes adrenal dysfunction directly and that hypersecretion of ACTH is a result of reduced corticosterone in rdw rats.

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The most common approach to analyzing the static morphology of mitochondria involves staining by antibodies or fluorescent dyes specific for mitochondrial components. In this study, we present a new approach using transgenic (Tg) mice, mtGFP-Tg mice, which exclusively express EGFP in the mitochondrial matrix. This Tg strain enables the rapid and easy observation of mitochondria in many kinds of tissues of interest. Recently, many reports have indicated that mitochondrial abnormalities and disease phenotypes are closely associated. mtGFP-Tg mice will be very useful in demonstrating this association, via the use of hybrids of mtGFP-Tg mice and well-established model mice for human diseases.

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In the present paper, we introduce a transgenic mouse line whose sperm express green fluorescent protein (GFP) in their acrosome and red fluorescent protein (RFP) in their mitochondria [B6D2F1- Tg(CAG/su9-DsRed2, Acr3-EGFP)RBGS002Osb]. The dual fluorescent sperm showed normal fertilizing ability in both in vivo and in vitro fertilization and the sperm could be observed through uterine and oviductal walls when female reproductive tracts were dissected out and placed under excitation light. This characteristic could facilitate examination of sperm migration inside the female reproductive tract as well as facilitating in situ live imaging of the acrosome reaction, the details of which have remained elusive.

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In previous genome-wide QTL studies, Pbwg12 on mouse chromosome 12 was discovered in a gene pool of wild Mus musculus castaneus mice. Pbwg12 does not have a main effect but has an epistatic interaction effect on body weight after birth. In this study, we developed a congenic strain, named B6.Cg-Pbwg12, with an approximately 59-Mb wild-derived genomic region harboring Pbwg12, by recurrent backcrossing to C57BL/6J. A phenotypic comparison between B6.Cg-Pbwg12 and C57BL/6J revealed that Pbwg12 does not have any main effects on body weight at 1-10 weeks of age but has a main effect on body weight gain at 6-10 weeks. A new QTL with a male-specific effect on kidney weight was discovered within the introgressed region.

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