In order to establish a system to facilitate the systematic collection, preservation, and provision of laboratory rats (Rattus norvegicus) and their derivates, the National BioResource Project-Rat (NBRP-Rat) was launched in July 2002. By the end of 2008, more than 500 rat strains had been collected and preserved as live animals, embryos, or sperm. These rat resources are supplied to biomedical scientists in Japan as well as in other countries. This review article introduces NBRP-Rat and highlights the phenome project, recombinant inbred strains, BAC clone libraries, and the ENU-mutant archive, named the Kyoto University Rat Mutant Archive (KURMA). The future direction of rat resources are also discussed.

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The Center for Animal Resources and Development (CARD), Institute of Resource Development and Analysis, Kumamoto University was established in 1998 based on recommendations published in the report "Preservation, Supply and Development of Genetically Engineered Animals" by the Ministry of Education, Culture, Sports, Science and Technology. We provide a comprehensive and integrated set of research services designed for the mouse-based biological research community. All services are conducted in accordance with the highest standards of animal health and genetic quality and are delivered to meet researcher's research goals. To promote biological sciences worldwide, we produce genetically engineered mice and exchangeable gene trap ES clones, cryopreserve mouse embryos and sperm, supply these resources, organize training courses to educate people, and form a hub of the domestic and international networks of both mutagenesis and resource centers. Up to now, we have produced more than 600 genetically engineered mouse strains and have more than 1,100 strains and stocks of mice for supply to the scientific community. More than 150 studies using genetically engineered mice produced or supplied by CARD have been published so far. As a founding member of the Federation of International Mouse Resources, the Asian Mouse Mutagenesis and Resource Association, and the International Gene Trap Consortium, we are contributing to the promotion of biological sciences in the world.

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The nematode C. elegans is a small and simple animal, which is easy to culture and store. Many detailed descriptions, biological resources, and methods for genetical and biochemical analyses have accumulated due to past research activities, and C. elegans is readily useful for molecular analyses with transgenic, RNAi, and mutants. Thus, C. elegans is an ideal model organism for detailed molecular analyses and functional genomics.

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The body and major organ weights of A/J-Chr 11^SM consomic mice were compared to those of the progenitor strains, A/J and SM/J. The weights of the body and organs, except for those of the brain and uterus, were significantly greater in A/J mice than in SM/J mice. However, those of consomic mice were highly variable. For example, the average body weight of consomic mice was significantly greater than that of SM/J mice and nearly equal to that of A/J mice. Chromosome 11 of SM/J mice induced various significant changes of the organ weights of A/J mice, especially those of the brain, lung, kidney, adrenal, and ovary, demonstrating the importance of this chromosome in determining the phenotypes.

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The Spontaneously Diabetic Torii (SDT) rat is a new model for non-obese type 2 diabetes. In the present study, we investigated changes in insulin secretion from the pancreas of male SDT rats aged 8, 16, and 24 weeks in order to analyze pancreatic function. An analysis of glucose-stimulated insulin secretion (GSIS) in isolated islets showed a marked reduction in insulin secretion in pre-diabetic 16-week-old SDT rats. When the islets were treated with tolbutamide or glucagon-like peptide-1 (7-36) amide (tGLP-1) in the presence of 11.2 mM glucose, however, insulin levels were restored to levels of normal rats. In vivo study, SDT rats exhibited a marked reduction in GSIS from 16 weeks of age. However, tolbutamide or JTP-76209, which is a novel dipeptidyl peptidase IV (DPP IV) inhibitor, increased insulin release after glucose loading and improved glucose tolerance. A marked reduction in GSIS was observed in pre-diabetic SDT rats and the reduction was improved by tolbutamide, tGLP-1, and the DPP IV inhibitor. Therefore, we concluded that the SDT rat is useful, as a model of non-obese insulin secretory disorder, for the analysis of the onset of type 2 diabetes and the development of antidiabetic agents.

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ICR-derived glomerulonephritis (ICGN) mice are a known inbred strain with hereditary nephrotic syndrome and are considered a good animal model of human idiopathic nephrotic syndrome. ICGN mice show proteinuria at a young age, and hypoalbuminemia, hyperlipidemia, anemia and edema accompanies their symptoms with aging. In addition, ICGN mice develop severe anemia with the progression of renal fibrosis similar to human chronic kidney disease (CKD). Recently, tissue transglutaminase (tTG) has been shown to be related to the renal fibrosis in several animal models and CKD patients. In the present study, we investigated the relationship between the progression of renal fibrosis and the localization of tTG in the kidneys using histochemistry and image analysis. Male ICGN mice aged 26-43 weeks were used. They were divided into two groups of early and terminal stages of renal fibrosis, based on plasma levels of blood urea nitrogen (BUN). Normal ICR males aged 11 weeks were used as a control group. tTG was localized to the interstitium in the normal ICR mice. In the early stage of renal fibrosis, the localization of tTG increased in renal tubules showing luminal dilation, as well as in the interstitium; however, the amount of tubular and interstitial tTG decreased in the late stage. In the glomeruli, tTG-immunoreactivity decreased in the late stage of renal fibrosis, despite the progression of glomerular sclerosis. The results suggest that ε(γ-glutamyl) lysine cross-linking is not directly related to the progression of renal fibrosis in ICGN mice.

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Spontaneously diabetic Torii (SDT) rats were established from Sprague-Dawley (SD) rat and are used as an animal model of type 2 diabetes mellitus. In the present study, the mechanism of the development of injury in the pancreas of these rats was examined focusing on the role of monocytes/macrophages. The number of lymphocytes and monocytes in the circulation of SDT rats increased with age, reaching a plateau at around 9 weeks of age and remaining at that level thereafter. The number of leukocytes in SDT rats was almost twice that of wild-type SD rats. Serum IL-18 levels began to increase at 8 weeks of age, forming a prominent peak at 9 weeks of age. In parallel with this, serum levels of NO₂/NO₃ showed an abrupt rise and decline. Spleen cells prepared from 9-week-old SDT rats expressed high levels of IFN-γ in response to IL-18, while those from 9-week-old wild-type SD rats did not. Immunohistochemical analysis revealed marked infiltration of CD68⁺ cells in the islets of SDT rats. Treatment of SDT rats with Cl₂MDP-liposomes reduced the number of monocytes as well as levels of NO₂/NO₃ in the circulation. Consistent with this, the number of infiltrated CD68⁺ cells in the islets was reduced in SDT rats treated with Cl₂MDP-liposomes. These results suggest that macrophages are involved in pancreatic islet injury in SDT rats through excess production of NO induced by IL-18 which increases transitorily at around 9 weeks of age.

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A large number of genetically modified mouse strains have been produced in recent years. Sperm cryopreservation is the most effective means of preserving these valuable strains, most of which have a C57BL/6 genetic background. However, the fertilization efficiency of sperm from several cryopreserved strains, including C57BL/6, is quite low. While new and improved methods of cryopreservation have been developed, the majority of sperm stocks have already been cryopreserved using traditional methods, such as storage in 18% raffinose and 3% skim milk (R18S3). Therefore, new thawing methods for these frozen stocks are needed. We have developed a new thawing method that involves selective collection of motile sperm and a preincubation medium that enhances capacitation. Motile sperm are selected simply by collecting a sample from the center of a dish, and capacitation is induced by the addition of methyl-beta-cyclodextrin, D-penicillamine, sodium citrate, and hypotaurine to modified Tyrode's solution. The fertilization rate of sperm prepared using this method was increased significantly compared to that of sperm thawed using the traditional method (63.9 vs 16.5%, P<0.01). These results demonstrate that this new in vitro fertilization method is an effective means of reviving C57BL/6 sperm cryopreserved in R18S3.

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Newborn neurons are continuously produced in the hippocampus, which may be involved in several cognitive functions, including learning and memory, throughout life. However, both hippocampus-dependent cognitive functions and the level of adult neurogenesis are gradually attenuated as aging progresses. Few studies have explored the relationship between adult neurogenesis and cognitive functions, especially in primates. In this study, we evaluated learning performance and hippocampal neurogenesis utilizing young and aged cynomolgus monkeys. Significant attenuations in learning performance and adult neurogenesis were detected in aged monkeys. Interestingly, there was a positive correlation between learning performance and the level of neurogenesis. Our findings suggest that cognitive functions and adult neurogenesis may have some interdependent relationships during aging.

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The WBN/Kob rat strain is a hereditary animal model of chronic pancreatitis and diabetes mellitus. The major WBN/Kob loci for pancreatitis (Pdwk1 and Pdwk2) are located on chromosomes 7 and X, respectively. In this study, polymorphisms were sought for candidate genes in the Pdwk1 and Pdwk2 regions. Nucleotide polymorphisms were found in 14 candidate genes examined in the Pdwk1 region. These polymorphisms were not associated with functional changes, and hence were unlikely to be a cause of pancreatitis. Seven nucleotide polymorphisms in three candidate genes, Rac2, Grap2, and Xpnpep3, located within a 3.3-Mb region were not found in 14 other inbred rat strains. These results suggest that WBN/Kob has a unique haplotype block in the chromosomal region contatining Pdwk1.

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Zona incision using a piezo-micromanipulator (ZIP) has been demonstrated to be effective for in vitro fertilization (IVF) using cryopreserved C57BL/6 spermatozoa. In this study, ZIP oocytes inseminated with frozen-thawed genetically modified C57BL/6J or FVB mice spermatozoa (21 lines) showed fertilization rates of 22-75% and live fetus rates of 8-49%. In 6 of the lines, the fertilization rates for oocytes compared with ZIP (42-75%) were significantly higher than that of nontreated oocytes (0-50%). Using only 90 oocytes for IVF with ZIP, 5 breeding pairs were produced from cryopreserved genetically modified mice spermatozoa. Our results indicate that application of the ZIP technique is effective for IVF using cryopreserved genetically modified mouse spermatozoa.

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Genetic alterations in the gene for ATP-binding cassette, sub-family B (MDR/TAP), member 1A (ABCB1A) determine susceptibility to colitis in mice and humans. We investigated the influence of ABCB1A dysfunction on susceptibility to dextran sulfate sodium (DSS)-induced colitis by using Senescence-Accelerated Mouse (SAM) strains with a loss-of-function mutation in the Abcb1a gene (SAMR1, SAMP1, and SAMP6). Susceptibility to DSS colitis was different among SAM strains but on the whole was not different from other mouse strains with normal ABCB1A function. Thus, genetic factors other than loss of ABCB1A are more crucial in determining susceptibility to DSS colitis in SAM strains.

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As members of Western societies age, sexual function of older (elderly) individuals becomes an important issue, particularly for men. Specifically, copulatory behavior increases circulatory load, which may be related to reports of cardiac sudden death following ejaculation. To further examine this relationship, we compared heart rate (HR) before and after ejaculation in 48-week-old (aged) and 10-week-old (young) male rats. As compared with resting HR, HR after ejaculation was increased by 54.2 ± 3.5 and 41.7 ± 2.7%, respectively, among aged and young male rats. These values were significantly higher than baseline levels (P<0.01). The increases in HR at each time point during copulation were significantly higher in aged male rats than in young male rats (P<0.05 or P<0.01). We also studied decreases in HR following ejaculation and found that aged male rats had a significantly higher HR at 1 and 2 min after ejaculation than young rats (P<0.01). These results suggest that the circulatory load on the aged rat heart is greater than that on a young rat heart during copulatory behavior, especially at ejaculation. In addition, the decrease in HR in aged rats after ejaculation was more gradual than in young male rats. These results suggest that there is a higher risk of sudden cardiac death during sexual behavior in older males.

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Anti-inflammatory effects of an ethanol extract of Angelica gigas (EAG; 50, 160, or 500 mg/kg) were investigated in a carrageenan-induced air pouch inflammation model. Injection of 1 ml of carrageenan (1%) into mouse air pouches markedly increased the exudate volume and exudate albumin concentration, which were significantly attenuated by oral pretreatment with EAG. EAG also markedly reduced carrageenan-induced infiltrations of neutrophils, monocytes, and lymphocytes, but did not influence eosinophils or basophils. Carrageenan dramatically increased levels of tumor necrosis factor-α and interleukin-6, which might be derived from the infiltrated cells. It also elevated nitric oxide, and slightly increased prostaglandin E₂. EAG pretreatment significantly lowered tumor necrosis factor-α and nitric oxide, but did not alter interleukin-6 or prostaglandin E₂ levels. These results indicate that EAG attenuates some inflammatory responses by blocking the tumor necrosis factor-α-nitric oxide pathway, and that EAG could be a promising anti-inflammatory drug candidate for inflammatory diseases.

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Recent advances in the genetic manipulation of mice have enabled us to generate transgenic and knockout mice. However, it is not easy to maintain these genetically-modified mice with the high-quality necessary to meet both scientific and legal requirements. RIKEN BRC has collected various transgenic, knockout, and conditional knockout mice. To examine the genetic modifications in these strains quickly and thoroughly, we established a simultaneous PCR test to detect multiple transgenes. We have called this, the "KO-survey". The PCR condition was optimized to detect neo, puro, pgk-neo, lacZ, and HSVtk-neo in set I, and hyg, IRES, cre, flp, and Gfp in set II. This "KO-survey" is useful for providing users with mouse strains of the highest genetic quality and accurate information on their genetic modifications.

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