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

我们正在使用逆转录病毒载体构建可用于 研究人类细胞突变机制的学科。在……下面 适当的感染条件可以隔离单拷贝 逆转录病毒载体的基因组整合，这是 诱变研究。这些研究中使用的逆转录病毒载体 携带并表达细菌gpt和neo基因。一个 Gpt基因和连锁的neo基因的适当整合 为细胞提供对霉酚酸和 氨基糖苷类，G418。我们已经评估了几个向量 构造来确定最佳表达式的要求 At基因和neo基因。我们找到了最佳的表达方式 Gpt基因受调控的构建 SV40启动子和neo基因的 5‘-LTR的控制。我们还在构建载体，允许 金属硫蛋白对gpt基因的差异调控 推动者。使用这些向量，我们正在生成 单拷贝gpt整合到人类细胞基因组中 线路，HT1080。HT1080细胞来源于人纤维肉瘤 并拥有特别稳定的核型，另一个重要的 诱变研究的要求。我们的目标是确定 基因组位置对自发和诱导的影响 人HT1080染色体整合gpt基因座的突变 细胞。将生成突变谱以进行比较分析 从各种HT1080派生的独立gpt集成。这样的一种 方法提供了一个独特的机会来评估“职位” 对使用相同靶基因(即gpt)的诱变的影响 整合在基因组的不同位置。从长远来看，这些 研究应为逆转录病毒的使用提供数据基础 载体在构建人类修复缺陷细胞系中的作用 对突变的机制研究很有用。

We are using retroviral vectors to construct cell lines useful fOr the study of mutational mechanisms in human cells. Under appropriate infection conditions one can isolate single copy genomic integrations of retroviral vectors, a requirement for mutagenesis studies. The retroviral vectors used in these studies carry and express both the bacterial gpt and neo genes. An appropriate integration of the gpt gene and the linked neo gene provides cellular resistance to both mycophenolic acid and the aminoglycoside, G418. We have evaluated several vector constructions to determine requirements for the optimal expression of both the at and neo genes. We find optimal expression in constructions in which the gpt gene is under the regulatory control of the SV40 promoter and the neo gene is under the regulatory control of the 5'-LTR. We are also constructing vectors that allow differential regulation of the gpt gene using the metallothionein promoter. Using these vectors, we are in the process of generating single copy gpt integrations into the genome of the human cell line, HT1080. HT1080 cells are derived from a human fibrosarcoma and have a particularly stable karyotype, another important requirement for mutagenesis studies. Our goal is to determine the effect of genomic position on both spontaneous and induced mutations at a chromosomally integrated gpt locus in human HT1080 cells. Mutant spectra will be generated for comparative analysis from various HT1080 derived independent gpt integrations. Such an approach provides a unique opportunity to evaluate "position effects" on mutagenesis using the same target gene (i.e., gpt) integrated at various sites in the genome. Long term, these studies should provide a data base for the use of retroviral vectors in the construction of human repair-deficient cell lines useful for mechanistic studies of mutations.