THE STUDY OF HOMOLOGOUS RECOMBINATION AND GENE REPAIR IN MOUSE SOMATIC CELLS.

本研究的主要目标是建立一种灵敏的检测染色体重组的方法，并在分子水平上分析重组机制。通过使用基因靶向建立在染色体11上的p53基因座处携带neo-tk盒的小鼠胚胎干（ES）细胞系（Gondo等人，Biochem.Bioprotein.Res.Commun.，202：830- 837，1994）。因此，这些ES细胞系是在p53基因座处携带野生型（W）和neo-tk（T）等位基因的杂合子。当W/T杂合子细胞用GANC处理时，检测到重组修复基因型，即W/W纯合子。反过来，T/T纯合子也分离出高浓度的G418。为了研究重组修复区及其周围的分子遗传学变化，我们还利用CBAXC 57 BL/6 F1小鼠的TT 2 ES细胞建立了W/T杂合子。初步研究表明，大约一半的11号染色体标记是异质的。 ...更多信息 TT 2中的卵合子。利用11号染色体上的多态性标记和鉴定的W/T到W/W或T/T的重组体，可以分析重组修复是否表现为1）狭义的基因转换，2）两个同源体之间的有丝分裂交换，3）单亲二体性不规则染色体分离。我们还开发了一个新的标记基因Eco-gpt，除了HSV-tk以增强敏感性以及减少选择系统的劳动密集型任务。我们已经建立了携带neo-gpt盒（G）等位基因的ES系，并确认基因型为W/G杂合子（Ito，Gondo et al.，正在筹备中）。通过6-tg筛选，从W/G ES细胞中分离出W/W纯合子。由于具有HSV-tk和Eco-gpt两个独特的负选择标记基因，因此构建T/G杂合子是可行的。例如，neo-tk整合到一个等位基因和嘌呤霉素13 R-gpt整合到另一个等位基因应该提供这样的杂合子。HSV-tk和Eco-gpt的标记可应用于哺乳动物基因组的任何部分，包括人类。因此，通过使用已建立的系统，同源重组现在可以在分子水平上进行研究，以阐明任何基因和物种的酶促过程。少

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 … More 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