Search

Almost 10,000 single nucleotide polymorphisms (SNPs) had been identified in the RT1 complex, the major histocompatibility complex of the rat, but less than approximately 0.5% have been characterized. In the context of the incomplete characterization of most SNPs, simple sequence length polymorphism (SSLP) marker development is still valuable for understanding the involvement of genes in the RT1 in controlling disease susceptibility, since SSLPs are user-friendly and cost-effective genetic markers in rat genome analysis. In this study, we developed a set of 67 SSLP markers, including 57 novel markers, to cover the entire RT1 complex and then created genetic profiles across 67 rat strains. These markers are located almost every 50 kb in the RT1 complex and show comparable polymorphism; the average number of alleles was 8.04 +/- 3.44 and the average polymorphic rate was 71 +/- 23%. Interestingly, markers failing to amplify polymerase chain reaction products were highly observed in all strains except for BN/SsNHsd, which suggests the existence of highly variable genomic sequences or genomic rearrangements in the RT1 region across rat strains. Based on the phylogenic tree and individual genotyping data, we identified 28 SSLP marker haplotypes in the RT1 region that roughly consisted of three genomic regions. These findings provided new insight into the genomic organization of the RT1 complex and we recognized the need of additional RT1 genome sequences in different strains. Owing to the accuracy and ease of determination, PCR-based SSLP genotyping could replace serological typing in genetic analyses and characterization of rat major histocompatibility.

Japan SLC was commissioned by the Council for SAM Research to produce and market in Japan and South Korea five strains (SAMP1, SAMP6, SAMP8, SAMP10 and SAMR1) of Senescence-Accelerated Mouse (SAM) in the summer of 2002. Japan SLC undertook microbiological control and genetic monitoring of these five strains and at the same time maintained and produced them under specific pathogen-free (SPF) conditions in a uniformly controlled breeding environment for market. The company used the basic “traffic-light-system” for mass production of SAM mice in their actual maintenance and production. In the selection of maintenance lines, by checking the specific biological characteristics and pathological phenotype of each strain in addition to the system, the principle in selection was to be able to supply mice of high reproducibility with the phenotypical characteristics of each strain.

The congenitally athymic nude rat is used for studying cancer and transplantation owing to its hairlessness and T-cell defective function caused by the Foxn1(rnu) gene. However, NK cell activity of the nude rat is markedly increased. It is known that NK cells play a major role in rejection of xenografts and in cytotoxicity against tumor cells. Thus, the athymic nude rat with impaired NK cell activity should be a useful model for extensive studies. The DA-Lyst(bg)/Lyst(bg) rat, a model for human Chediak-Higashi syndrome (CHS) is characterized by diluted-coat color and impairment of NK cell activity. We planned to establish a combined immunodeficient double mutant rat introgressed with the Foxn1(rnu) and Lyst(bg) genes and a set of congenic strains having an identical genetic backgrounds simultaneously. Based on the phenotypic and genetic characteristics of the parental rat strains, the new strains were produced using continuous backcross and diagnosis with molecular genetic techniques. Each disease gene was diagnosed with PCR-RFLP or the long-nested PCR method. Furthermore, we used a marker-assisted congenic strategy based on scanning the genetic backgrounds of the parental rats with 461 rat microsatellite markers. We think that the newly established DA/Slc-Foxn1(rnu)/Foxn1(rnu) Lyst(bg)/Lyst(bg) double mutant will be useful as a severe disease model for human CHS, and the set of DA/Slc-Foxn1(rnu) Lyst(bg) congenic strains which have impaired NK cell activity and/or defective T cell function should be useful for studying in cancer research, xenotransplantation, immune function and other wide-ranging studies.

Two spontaneous mutant beige rats, with phenotypes resembling human Chediak- Higashi syndrome (CHS), were found independently in two inbred strains. Both beige mutations were identified to be recessive alleles in the Lyst locus on rat chromosome 17 and the alleles were denoted Lyst(bg) and Lyst(bg-Kyo). As it is almost impossible to discriminate these mutations phenotypically, we developed an allele-specific genotyping method for the Lyst gene. The nested PCR amplification was followed by restriction fragment length polymorphism (RFLP) analysis. By this method, we could discriminate the mutant Lyst(bg), Lyst(bg-Kyo) alleles, and the normal Lyst allele, easily and accurately.

Clarifying the participation of oxidative stress among possible contributing factors in potassium bromate (KBrO(3))-induced carcinogenesis is of importance from the perspective of human health protection. In the present study, utilizing the antioxidative effects of alpha-tocopherol (alpha-TP) or sodium ascorbic acid (SAA) to attenuate oxidative stress, alterations in bromodeoxyuridine labeling indices (BrdU-LIs) and reporter gene mutations in kidneys of male and female gpt delta rats given KBrO(3) were examined. Five male and female gpt delta rats in each group were given KBrO(3) at a concentration of 500ppm in the drinking water for 9 weeks, with 1% of alpha-TP or SAA administered in the diet from 1 week prior to the KBrO(3) treatment until the end of the experiment. Increases in 8-hydroxydeoxyguanosine levels in kidney DNA of both sexes of rats given KBrO(3) were significantly inhibited by SAA, but not alpha-TP. While BrdU-LIs in the proximal tubules of female rats were also significantly reduced by SAA, those in the males and gpt mutant frequencies in kidney DNA of both sexes were not affected by SAA or alpha-TP. Immunohistochemical and Western blot analyses for alpha(2u)-globulin strongly suggested that induction of cell proliferation observed in the males might primarily result from accumulation of this protein, independent of oxidative stress. The overall data indicated that while oxidative stress well correlates with induction of cell proliferation in females, its role in males and in generation of in vivo mutagenicity by KBrO(3) in both sexes is limited.

A new method of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was developed for genetic typing of a point mutation of the Bruton's tyrosine kinase (Btk) gene in CBA/N mice bearing an X-linked recessive immunodeficiency (xid). Since restriction site useful for RFLP analysis does not exist in the spontaneous mutant Btk(xid) locus, an artificial restriction site was introduced by PCR amplification with a modified primer. The five genotypes of the Btk locus (Btk(xid)/ Btk(xid), Btk(xid) /Btk+ and Btk+/Btk+ females and Btk(xid)/Btk(null) and Btk+/Btk(null) males) could be distinguished by three patterns clearly and easily. This PCR-RFLP analytic method will be useful as a tool in the production of congenic mice and mice with multiple immunodeficient genes.

A new polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was developed for genetic typing of the mouse Tnfsf6gld mutation. An artificial restriction site was introduced to the mouse Tnfsf6gld mutation by PCR amplification using a modified primer. The three genotypes of the Tnfsf6 locus (Tnfsf6gld/Tnfsf6gld, Tnfsf6gld/+, and +Tnfsf6-gld/+Tnfsf6-gld) could be distinguished clearly and easily. This PCR-RFLP analysis was found to be useful for the identification of the mouse Tnfsf6gld mutation.

Ion beam etching (IBE) of GaAs with a source gas of Cl2 or Ar was carried out at a low energy ranging from 5 to 130 eV by using an electron-beam-excited-plasma system, and residual defect centers were investigated by means of deep-level transient spectroscopy (DLTS). Foul kinds of defect centers labeled L1 L2, L3, L5 with activation energies of 0.31, 0.45, 0.58, 0.48 eV, respectively, were resolved. Three of them were associated with damages induced by IBE and it was found that they have different thresholds for their generation, i.e., 60, 40 and 20 eV for L1, L2 and L3, respectively. It is necessary to reduce ion energy to less than 20 eV for perfect damage-free IBE of GaAs.