DETECTION OF MUTATION BY TRANSGENE REVERSION IN MICE

小鼠中转基因逆转突变的检测

• 批准号: 6346149
• 负责人: PETER J. STAMBROOK
• 金额: $ 13.47万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6346149
• 关键词: adenine phosphoribosyltransferase; autoradiography; biphenyl compounds; cell type; chemical carcinogen; electroporation; embryonic stem cell; environmental toxicology; gene expression; gene frequency; gene mutation; gene targeting; genetic markers; genetic promoter element; genetic recombination; genetically modified animals; homozygote; laboratory mouse; microinjections; model design /development; mutagen testing; nitrosourea; point mutation; reporter genes; site directed mutagenesis

The purpose of this project is to produce a set of mice that utilize an endogenous gene as a reporter of mutagenic activity and to establish the frequency of spontaneous and induced mutation at athe single cell level. The reporter gene is that which encodes adenine phosphoribosyltransferase (APRT), a purine salvage enzyme that catalyzes the conversion of adenine to AMP. Mice that lack the enzyme are viable. When APRT deficient cells are treated with labeled adenine, the adenine will enter and exit the cells without impediment. However, any cell, and its progeny, that reverts to Aprt+, will phosphoribosylate labeled adenine to AMP and thereby entrap athe labeled product ina the cell. The labeled adenine will be sequestered and will accumulate intracellularly, and will be incorporated into nucleic acid. Thus, the Aprt+ cells will be selectively labeled. Using targeted homologous recombination in embryonic stem (ES) cells, the wildtype Aprt gene will be replaced with a set of mutant alleles containing all combinations of transitions and transversions at a splice acceptor site, as well as a frameshift mutation in exon 1. Mice will be produce with each of these mutant alleles and will be tested for the frequency of spontaneous mutation in each of multiple organs. This will be accomplished by injecting mice IP with 14C-adenine, allowing 48 hours to 96 hours for the labeled adenine to be cleared in the urine, and examining tissue sections of selected organs for radioactive cells, or patches of cells by autoradiography. Having determined the spontaneous level of mutation at the cellular level, mice will be challenged with two model mutagens, one of which is a direct-acting mutagen (N-ethyl-N-nitrosourea) and the other a mutagen requiring metabolic activation (4-aminobiphenyl). The impact of each of these mutagens/carcinogens upon mutation frequency in different tissues and organs will e assessed, as will the cell type(s) most affected. The mutant Aprt alleles also will be crossed into TGFbeta-1 null and PMS2 null genetic backgrounds to establish whether or not the absence of these encoded activities affects wither spontaneous or induced mutation levels. The question also will be posed whether tissues and organs that sustain the most mutations are those that develop tumors with the highest frequency.

该项目的目的是生产一组利用一个小鼠 内源基因作为诱变活性的报道者，并建立 自发和诱导的单细胞水平突变的频率。 记者基因是编码腺嘌呤磷酸贝糖基转移酶的基因 （APRT），一种嘌呤挽救酶，可催化腺嘌呤转化为 放大器。 缺乏酶的小鼠是可行的。 当APRT缺乏细胞为 用标记的腺嘌呤处理，腺嘌呤将进入并退出细胞 没有障碍。 但是，任何细胞及其后代都恢复了 APRT+，将磷酸糖基酯标记为AMP，从而捕集 标记的产品Ina。 标记的腺嘌呤将被隔离 并会在细胞内积累，并将掺入核酸中 酸。 因此，将选择性地标记APRT+细胞。 使用目标 胚胎茎（ES）细胞中的同源重组，野生型APRT 基因将被一组包含全部的突变等位基因代替 剪接受体站点的过渡和转移的组合， 以及外显子1中的移码突变。每个小鼠都会与每个小鼠一起产生 这些突变等位基因，并将测试自发的频率 在每个器官中的每个器官中突变。 这将通过 将小鼠IP注入14c-腺嘌呤，可让48小时至96小时 标记为要在尿液中清除的腺嘌呤，并检查组织切片 用于放射性细胞的选定器官，或通过 放射自显影。 确定了自发的突变水平 细胞水平，小鼠将被两个模型诱变剂挑战，其中之一 这是一种直接作用的诱变剂（N-乙基-N-硝化诱变），另一个是A 需要代谢活化的诱变剂（4-氨基苯基）。 的影响 这些诱变剂/致癌物在突变频率不同 组织和器官将评估，受影响最大的细胞类型。 突变的APRT等位基因也将越过TGFBETA-1 NULL和PMS2 零遗传背景以确定是否缺乏 编码活动会影响自发或诱发的突变水平。 问题还将提出维持的组织和器官 大多数突变是发展频率最高的肿瘤的突变。