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A MUTATION DETECTION SYSTEM FOR MAMMALS AT INDIVIDUAL LEVEL

哺乳动物个体水平的突变检测系统

基本信息

批准号：
63580168
负责人：
INOUE Tadashi
金额：
$ 1.22万
依托单位：
Nihon University, College of Agriculture and veterinary Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63580168/
关键词：
Mutation Mouse Lambda phage 検出系試験管内パッケージ

项目摘要

Quantitative assay for mutation induction in mammals at individual level is of importance to evaluate the effect on humans of environmental mutageiis and carcidogens as well as genotoxic agents, which have been recently used in modern living. Over hundreds thousands of mice are required, however, to carry out experiments for quantitation of mutation frequencies. We therefore tried to develop an assay system in which only a small number of animals are required for the detection of mutation at the individual level.The system has been constructed by combination of phage genetics and transgenic mouse system. As a marker gene, supF gene of E. coli was introduced into lambda phage EMBL3. When mixed with their proteins, the DNA of lambda phage can be packaged in vitro to yield lambda phage particles. Mutation in supF gene in the lambda phages is easily detected by using a proper E. coli as an indicator cells.Purified DNA of the lambda phage containing supF was injected into fertilized eggs of mice to establish a transgenic mouse. Presence of the transgene in the developed mice was examined by southern analysis of the DNA extracted from tails using supF gene as a probe. To date, we have obtained several strains of transgenic mice. We are now examining the organization of the transgene in the transgenic mice by restriction analysis. Once a strain with appropriate properties has established, DNA from sperms from the mice is subjected to the in vitro packaging system to analyze the mutation in supF gene occurred in vivo.

在个体水平上对哺乳动物进行诱变诱导的定量分析，对于评价环境诱变物、致癌物以及遗传毒性物质对人类的影响具有重要意义。然而，要进行定量突变频率的实验，需要数十万只老鼠。因此，我们试图开发一种检测系统，在这种系统中，只需要少量的动物来检测个体水平上的突变。该系统采用噬菌体遗传学与转基因小鼠系统相结合的方法构建。将大肠杆菌supF基因作为标记基因导入lambda噬菌体EMBL3。当与它们的蛋白质混合后，λ噬菌体的DNA可以在体外包装以产生λ噬菌体颗粒。利用合适的大肠杆菌作为指示细胞，很容易检测到λ噬菌体中supF基因的突变。将含有supF的噬菌体纯化DNA注射到小鼠受精卵中，建立转基因小鼠。利用supF基因作为探针，对从小鼠尾部提取的DNA进行southern分析，检测转基因在发育小鼠体内的存在。到目前为止，我们已经获得了几个转基因小鼠品系。我们现在正通过限制性内切分析来检查转基因小鼠的转基因组织。一旦建立了具有适当特性的菌株，从小鼠精子中提取DNA进行体外包装系统，以分析体内发生的supF基因突变。