GENE CONVERSION ASSOCIATED WITH HOMOLOGOUS RECOMBINATION AND DNA REPAIR OF THE MOUSE.

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Mouse embryonic stem (ES) cell lines that carried a neo-tk cassette at the p53 locus on the chromosome 11 were established by nsing gene targeting. These ES cell lines for the first time provided a system to investigate the phenomena with respect to the homologous recombination of chromosomes. It has been demonstrated that the method encompassing two steps of gene targeting universally enabled to replace a gene with any DNA sequences. During the process of the development of this method, gene conversions were observed as clones of totally unexpected types of pseudo-homologous recombinants. Approximately 25% of the p53 gene-targeted clones were pseudo-homologous recombinants. In addition, the recombination repair has been also recognized between the wild-type (W) and targeted-type (T) alleles in theW/T heterozygote. This recombination repair occurred to either direction consistently. These achievements were already published (Gondo, et al., Biochem.Biophys.Res.Commun., 1994)Three possib … More le models have been proposed for the molecular mechanism of the recombination repair. One is the gene conversion of the excision-repair type at the limited region of the p53 gene and its vicinities. The second feasible hypothesis is the mitotic recombination between the sister chromatids of the two homologues after S period of the cell cycle and subsequent segregation of the same alleles to the same daughter cells. The last model postulates no segregation of two sister chromatids of a homologue at mitosis resulting a gain of homozygosity of the entire chromosome as found in uniparental disomy. In order the examine the three working hypotheses, it is necessary to have a highly polymorphic ES cell line with molecular markers which allow us to distinguish those polymorphisms. Thus, one of such polymorphic ES cell line, TT2, was obtained from Dr.S.Aizawa (Kumamoto University). The TT2 ES cell line was established from a heterozygote of two inbred strains, CBA and C57BL/6. The gene targeting was performed again and 17 of the TT2 ES cell clones carrying the neotk cassette were newly established. The molecular markers on the chromosome 11 on which the p53 gene is were also investigated and 4 SSLP markers have been found so far to be polymorphic between CBA and C57BL/6.By using this system, it became plausible to study the molecular mechanisms of the homologous recombination at a defined region of the chromosomes in the mammalian genome. More polymorphic markers on the chromosome 11 have been currently under investigation. Less