|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1996 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1995 : ¥1,300,000 (Direct Cost : ¥1,300,000)
The major objective of this study is to establish a sensitive method of detecting chromosomal recombinations and to analyze recombinational mechanisms at the molecular level. Mouse embryonic stem (ES) cell lines that carried a neo-tk cassette at the p53 locus on the chromosome 11 were established by using gene targeting (Gondo et al., Biochem.Biophys.Res.Commun., 202 : 830-837,1994). These ES cell lines were, thus, heterozygotes carrying the wild-type (W) and neo-tk (T) alleles at the p53 locus. When W/T heterozygous cells were treated with GANC,recombinational repair genotype, namely, W/W homozygotes were detected. Reciprocally, T/T homozygotes were also isolated with a high concentration of G418. In order to investigate the molecular genetic changes at and around the recombinational repair region, we also established W/T heterozygotes by using TT2 ES cells which derived from a CBAXC57BL/6 F1 mouse. Preliminary studies indicated approximately a half of chromosome 11 markers were heter
ozygous in TT2. By using these polymorphic markers of chromosome 11 and identified recombinants of W/T to W/W or T/T,it is now possible to analyze whether the recombinational repair was manifested by 1) gene conversion with a narrow sense, 2) mitotic crossing-over between two homologous, or 3) uniparental disomy-type irregular chromosomal segregation.
We also developed a new marker gene, Eco-gpt, in addition to HSV-tk to enhance the sensitivity as well as to reduce the labor-intensive tasks of the selection system. We have established ES lines carrying neo-gpt cassette (G) allele and confirmed the genotypes were W/G heterozygotes (Ito, Gondo et al., in preparation). W/W homozygotes were also isolated from W/G ES cells by 6-tg selection. Having two distinctive negatively selectable marker genes like HSV-tk and Eco-gpt, it is plausible to construct T/G heterozygotes. For instance, neo-tk integration to one allele and puromycin^R-gpt integration to the other should provide such heterozygotes. It will provide a new tool of the selection for recombinational repair between two heterozygous homologues to both directions ; from G/T to T/T and to G/G.The tagging of HSV-tk and Eco-gpt should be applicable to any part of mammalian genome including human when a tissue culture system is available. Thus by using the established system homologous recombination is now feasible to be investigated at the molecular level to elucidate the enzymatic process at any genes and species. Less