USE OF RETROVIRAL VECTORS IN THE ANALYSIS OF MUTATIONS IN MAMMALIAN CELLS

哺乳动物细胞中诱导突变的DNA序列分析 由于缺乏系统， 分离的突变体的方便分析。 我们的方法是 构建旨在简化分离的细胞系， 在哺乳动物细胞中诱导的突变的表征。 为了产生可用于诱变研究的哺乳动物细胞系， 我们已经构建了几种携带gpt的逆转录病毒载体， 和其它选择标记。 这些载体可用于 感染多种哺乳动物细胞类型。 的整合 逆转录病毒序列随机出现，通常是单拷贝 在基因组中。 因此，任何两个孤立的 细胞克隆将携带插入的GPT基因， 基因组定位 因此，我们可以研究 使用相同靶基因进行诱变的基因组位置 (gpt)在多个站点集成。 我们有AS 52的数据 细胞系，表明缺失突变是受 靶基因的基因组位置;然而，没有数据 可用于评估基因组位置对 自发或诱导的点突变。 我们将利用 逆转录病毒载体分离人类和小鼠修复精通 具有单拷贝GPT整合的细胞克隆， 在GPT基因座的自发和诱导的点突变。 将分离突变DNA并测序以产生突变体 谱 所产生的光谱的比较将允许 评估基因组位置对 哺乳动物中产生的点突变的发生率和类型 细胞 这些数据应为使用 人修复缺陷细胞系，例如着色性干皮病， 共济失调性毛细血管扩张症、布卢姆综合征等，推导出 逆转录病毒载体在未来研究中的突变机制， 哺乳动物细胞

DNA sequence analysis of mutations induced in mammalian cells has been limited by the lack of systems available for the convenient analysis of isolated mutants. Our approach has been to construct cell lines designed to simplify the isolation and characterization of mutations induced in mammalian cells. To generate mammalian cell lines useful for mutagenesis studies, we have constructed several retroviral vectors that carry gpt and other selectable markers. These vectors can be used to infect a variety of mammalian cell types. The integration of retroviral sequences occurs randomly and usually in single copy in the genome. Therefore, it is unlikely that any two isolated cell clones will carry the gpt gene inserted at precisely the same genomic location. We can, therefore, study the effect of genomic position on mutagenesis using the same target gene (gpt) integrated at several sites. We have data with the AS52 cell line that suggests deletion mutations are influenced by the genomic position of the target gene; however, there are no data available to assess the influence of genomic position on either spontaneous or induced point mutations. We will utilize retroviral vectors to isolate human and mouse repair proficient cell clones with single copy gpt integrations and will generate both spontaneous and induced point mutations at the gpt locus. Mutant DNAs will be isolated and sequenced to generate mutant spectra. Comparison of the spectra generated will allow an assessment of the influence of genomic position on the occurrence and type of point mutations generated in mammalian cells. These data should provide a foundation for the use of human repair deficient cell lines, e.g. Xeroderma pigmentosum, Ataxia telangiectasia, Bloom's Syndrome, etc., derived using retroviral vectors in future studies of mutational mechanisms in mammalian cells.