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

小鼠体细胞同源重组和基因修复的研究。

• 批准号: 07640823
• 负责人: GONDO Yoichi
• 金额: $ 1.41万
• 依托单位: TOKAI UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07640823/
• 关键词: homologous recombination; somatic cells; gene conversion; mouse; tissue culture cells; DNA repair; molecular genetics; 高等動物; 分子遺伝子; ジーンターゲッテイング; マウスゲノム; 不等価組換え

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

本研究的主要目的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。利用基因打靶技术(Gondo等，BioChem.BiPhys.Res.Comm.，202：830-837,1994)建立了在11号染色体上的P53位点处携带neo-tk盒的小鼠胚胎干细胞(ES)细胞系。因此，这些ES细胞系是杂合子，在p53基因座上携带野生型(W)和neo-tk(T)等位基因。当用GANC处理W/T杂合子细胞时，检测到重组修复基因，即W/W纯合子。反过来，也用高浓度的G418分离出T/T纯合子。为了研究重组修复区域及其周围的分子遗传学变化，我们还利用来源于CBAXC57BL/6 F1小鼠的TT2 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heter…。TT2的受精卵更多。通过利用11号染色体的这些多态标记和鉴定W/T到W/W或T/T的重组子，现在可以分析重组修复是否表现为1)狭义的基因转换，2)两个同源的有丝分裂互换，或3)单亲二体类型的不规则染色体分离。除了HSV-tk，我们还开发了一个新的标记基因Eco-gpt，以提高选择系统的敏感性，并减少选择系统的劳动强度。我们已经建立了携带neo-gpt cassette(G)等位基因的ES系，并确认其为W/G杂合子(Ito，Gondo等人，正在准备中)。通过6-TG筛选从W/G ES细胞中分离出W/W纯合子。由于有两个独特的负选择标记基因HSV-tk和Eco-gpt，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，以及嘌呤霉素R-gpt整合到另一个等位基因，就应该提供这样的杂合子。这将为从G/T到T/T和G/G两个方向的杂合同源物之间的重组修复提供一个新的选择工具。当有组织培养系统时，HSV-tk和Eco-gpt的标记应该适用于包括人类在内的哺乳动物基因组的任何部分。因此，通过使用建立的系统，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过程。较少

项目成果

権藤洋一、勝木元也: "11.4 DNA変異を検出する方法." 講談社サイエンティフィク, 11 (1995)

Yoichi Gondo、Motoya Katsuki：“11.4 DNA 突变检测方法。”讲谈社科学，11 (1995)

Hideo Shibata, Kiyoshi Yoshino, Shoichi Sunahara, Yoichi Gondo, Motoya Katsuki, Takayuki Ueda, Mamoru Kamiya, Masami Muramatsu, Yasufumi Murakami, Iveta Kalcheva, Christoph Plass, Verne M.Chapman and Yoshihida Hayashizaki: "Inactive allele-specific methyl

Hideo Shibata、Kiyoshi Yoshino、Shoichi Sunahara、Yoichi Gondo、Motoya Katsuki、Takayuki Ueda、Mamoru Kamiya、Masami Muramatsu、Yasufumi Murakami、Iveta Kalcheva、Christoph Plass、Verne M.Chapman 和 Yoshihida Hayashizaki：“非活性等位基因特异性甲基

Yoichi Gondo and Motoya Katsuki: "Transgenic system for the genotoxicity test. (in Japanese)" The Tissue Culture. 22 (13). 522-525 (1996)

Yoichi Gondo 和 Motoya Katsuki：“遗传毒性测试的转基因系统。（日语）”组织培养。

T.Kitamoto,Y.Gondo,et al.: "Humanized prion protein knock-in by Creinduced site-specific recombination in the mouse." Biochem.Biophys.Res.Commun.222. 742-747 (1996)

T.Kitamoto、Y.Gondo 等人：“在小鼠中通过 Creinduced 位点特异性重组实现人源化朊病毒蛋白敲入。”

Yoichi Gondo, Yoshiyuki Shioyama, Kazuki Nakao, Motoya Katsuki: "A novel positive detection system of in vivo mutations in rpsL (strA) transgenic mice." Mutation Res.360. 1-14 (1996)

Yoichi Gondo、Yoshiyuki Shioyama、Kazuki Nakao、Motoya Katsuki：“一种新型的 rpsL (strA) 转基因小鼠体内突变阳性检测系统。”