Studies of Somatic Mutation Frequencies and Spectra with a Sensitive and Efficient Detection Method by Using Transgenic Mice.

利用转基因小鼠以灵敏、高效的检测方法研究体细胞突变频率和谱。

• 批准号: 07554043
• 负责人: GONDO Yoichi
• 金额: $ 13.06万
• 依托单位: TOKAI UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07554043/
• 关键词: Transgenic mouse; Mutation ; in vivo; rpsL gene; Tumorigenicity screening; Genotoxicity test; Mutagen; Mutagenicity test; Shuttle vector; ypsL; 突然変異検定; 分子遺伝学; マウス; rpsL遺伝子

We have developed a transgenic system to detect somatic mutations in vivo by using the E.coli rpsL gene as a positively selectable marker. In addition to the pML4 shuttle vector, a newly constructed pSSW was also introduced into mice. As results ; 1) the background mutant frequency was reduced to 1x10^<-5>,2) the efficiency of the detection increased 3-fold, 3) methylated shuttle vectors were rescued in E.coli, 4) the mode of mutations was the indistinguishable between two shuttle systems. Mutagens, i.e., MNU,MeIQx, AOM,AFBI and Trp-P-2 were examined so far. Effects of antimutagenicity of chlorophirin and EGCG were also studied. The mutagenicity of rpsL was extensively analyzed in the E.coli system in terms of mutators of hosts and/or hot spot sequences in the rpsL gene. The comparison was made with newly developed positive selection systems with the gpt and cII transgenic mice in addition to MutaMouse and BigBlue. The delayd radiation effects were recognized in mice as well as Drosophila, which are the candidate systems for the future research. Progenies were obtained from the mating of pML4-or pSSW-transgenic mice to the mice deficient for the XPA or MLH1 gene. The mutation spectra studies suggested the possibility to distinguish the effect of mutagenicity from the effect of cytotoxicity of an administered agent.

我们开发了一种转基因系统，通过使用大肠杆菌 rpsL 基因作为正选择标记来检测体内体细胞突变。除了pML4穿梭载体外，还将新构建的pSSW引入小鼠体内。作为结果； 1)背景突变频率降低至1x10^-5>，2)检测效率提高3倍，3)甲基化穿梭载体在大肠杆菌中被拯救，4)两个穿梭系统之间的突变模式无法区分。目前已检测了诱变剂，即 MNU、MeIQx、AOM、AFBI 和 Trp-P-2。还研究了叶绿素和 EGCG 的抗突变作用。在大肠杆菌系统中，根据宿主突变体和/或 rpsL 基因中的热点序列对 rpsL 的致突变性进行了广泛分析。除了 MutaMouse 和 BigBlue 之外，还与新开发的正选择系统与 gpt 和 cII 转基因小鼠进行了比较。延迟辐射效应在小鼠和果蝇中得到了证实，它们是未来研究的候选系统。 pML4 或 pSSW 转基因小鼠与缺乏 XPA 或 MLH1 基因的小鼠交配获得后代。突变谱研究表明有可能区分所施用药物的致突变性影响和细胞毒性影响。

项目成果

H.Nakane: "High incidence of ultraviolet-B-or chemical-carcinogen-induced skin tumours in mice lacking the xeroderma pigmentosum proup A gene." Nature. 337. 165-168 (1995)

H.Nakane：“缺乏色素性干皮病 proup A 基因的小鼠中，紫外线 B 族或化学致癌物诱发的皮肤肿瘤发生率很高。”

T.Kitamoto: "Humanized prion protein knock-in by Cre-induced site-specific recombination in the mouse." Biochemical and Biophysical Research Communication. 222. 742-747 (1996)

T.Kitamoto：“通过 Cre 诱导的小鼠位点特异性重组实现人源化朊病毒蛋白敲入。”

O.Niwa: "Induction of a germline mutation at a hypervariable mouse minisatellite locus by 25 2-Cf radiation." Jounal of Radiation Research. 37. 217-224 (1996)

O.Niwa：“通过 25 2-Cf 辐射在高变小鼠小卫星位点诱导种系突变。”

Y.Gondo: "Analysis of genetic instabilities of RS447 megasatellite DNA region in human 4p15." American Journal of Human Genetics. 61. A391- (1997)

Y.Gondo：“人类 4p15 中 RS447 大卫星 DNA 区域的遗传不稳定性分析。”

権藤 洋一: "「抗変異原・抗発がん物質とその検索法」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：“‘抗诱变剂/抗致癌物质及其搜索方法’11.4 DNA突变检测方法，讲谈社科学，11（1995）”