| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2002: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2001: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
The thymoma-prone BUF/Mna (B) rat is a useful model for human thymoma. At pre-thymoma age, B rats have extremely large thymuses when compared with those of rats of the other strains, suggesting the presence of genes that regulate the thymus enlargement. Linkage study showed the significant associations between thytnus size and polymorphic markers on chromosomes (Chrs) 1 and 13, suggesting the presence of two genes, Ten 1 and Ten 2, which regulate the thymus enlargement. To further characterize the precise position of Ten 1, 34 and 37 microsatellite markers, mat have a polymorphism between the B and WKY (W) and between the B and MITE (M) strains, were used for linkage analysis of thymus enlargement in 105 (WBF1 x B) backcross (BC) and 78 (B x BMF1) BC rats, respectively. The markers, D1Rat168. D1Rat112. D1Rat323, D1Got186, D1Got187, D1Got188, each gave a peak LOD score of 10.68 for linkage to the thymus ratio in (WBF1 x B) BC rats. The D1Rat168, D1Rat197, D1Got186 and D1 Got188 makers e
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ach gave a peak LOD score of 7.82 in (B x BMF1) BC rats. The two LOD score peaks were coincident in the position of the rat genetic map. All the markers are located in the region between Igf2 and D1Mgh11. Synteny of the region is conserved with human 11q15.5 and the distal end of mouse Chr 7 or with human 11q13 and the proximal end of mouse Chr 19. Phenotype-directed representational difference analysis (RDA) was used to isolate 28 polymorphic markers tightly linked to the thymus enlargement loci. These markers were polymorphic between W and B strains. Eight of the markers were shown to be linked to Ten 1, and one to Ten 2. One of the 8 markers linked to Ten 1 demonstrated no recombination in 18 rats with high thymus ratios. Using several markers as probes, a cosmid genomic library constructed from SD rats with normal thymus ratio were screened with blot hybridization method. One genomic clone positive for one of the polymorphic markers was partially sequenced at both ends of the insert PCR amplified fragments derived from the each ends were used to screen the library for expanding the cosmid contig. Four genomic clones covering about 120 kb were obtained and characterization of these clones by sequences and by fluorescent in situ hybridization (FISH) are now in progress. Since a cosmid clone showed deletion during expansion, screening of high density filter (macroarray) which contained 3- to 4-fold coverage genomic library constructed in BAG library is also conducted. Less
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