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

哺乳动物细胞诱变的DNA序列分析 由于缺乏可用的系统， 方便地分析分离的突变体。我们的方法一直是 构建旨在简化分离和分离的细胞系 在哺乳动物细胞中诱导突变的特征。 为了产生对突变研究有用的哺乳动物细胞系， 我们已经构建了几个携带gpt的逆转录病毒载体。 和其他可选择的标记。这些向量可用于 感染多种哺乳动物细胞类型。整合了 逆转录病毒序列随机出现，通常以单拷贝形式出现 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 基因组位置。因此，我们可以研究 同一靶基因诱变中的基因组位置 (GPT)在多个地点整合。我们有AS52的数据 提示缺失突变的细胞系受 目标基因的基因组位置；然而，没有数据 可用于评估基因组位置对其中一个的影响 自发的或诱发的点突变。我们将利用 逆转录病毒载体分离人和小鼠修复熟练者 具有单拷贝gpt集成的细胞克隆，并将生成 Gpt基因座的自发和诱导点突变。 突变的DNA将被分离和测序以产生突变 光谱。对产生的光谱进行比较将允许 基因组位置对基因定位影响的评估 哺乳动物产生的点突变的发生和类型 细胞。这些数据应该为使用 人类修复缺陷细胞系，例如着色性干皮病， 共济失调、毛细血管扩张、布卢姆综合征等 逆转录病毒载体在未来突变机制研究中的应用 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.