EXPRESSION OF GENETIC VARIATION IN CULTURED CELLS

这项研究的目标是确定在何种程度上 突变外事件，如有丝分裂重组，基因 转换、基因失活、多位点缺失和染色体 非分离或染色体重排，都参与了 隐性突变在培养的哺乳动物细胞中的表达。我们 建议开发和利用几个模型系统，这些系统将 促进对这些遗传过程的研究 分子水平。我们的最终目标是清楚地了解 参与细胞过程和机制的产生和 哺乳动物体细胞中可遗传变异的表达。一个 系统便于检测和分析目标 同源重组事件将使用一系列 CHO APRT缺失或插入突变体。这个系统将是 用于研究转基因OR的靶向重组 内源性CHO aprt基因序列的显微注射 位置；确定目标的相对频率 整合和基因转化事件，影响大小不一 靶基因序列中的缺失或插入 重组频率，以及转录的基因是否更好 定向同源重组的靶点比非 转录的基因。另一种系统，使用CHO细胞杂交 在不同的、非重叠的APRT删除之间构建 突变，将被用来测试自发或诱导频率 内源APRT的等位基因间有丝分裂重组 它们正常染色体环境中的等位基因。第三个系统 将检查纯合子或半合子的生成 由遗传事件引起的培养细胞的隐性突变 有丝分裂重组、染色体不分离或多位点 在传统CHO和V-79中无法检测到的缺失 诱变试验。其他研究将重点放在 染色体环境对基因相对易感性的影响 突变、缺失或重组。

The objective of this research is to determine the extent to which extramutational events, such as mitotic recombination, gene conversion, gene inactivation, multilocus deletions, and chromosome non-disjunction or chromosomal rearrangements, are involved in the expression of recessive mutations in cultured mammalian cells. We propose to develop and utilize several model systems that will facilitate investigation of these genetic processes at the molecular level. Our ultimate goal is a clear understanding of the cellular processes and mechanisms involved in the generation and expression of heritable variation in mammalian somatic cells. A system to facilitate the detection and analysis of targeted homologous recombination events will be developed using a series of CHO APRT deletion or insertion mutants. This system will be used to study targeted recombination of transfected or microinjected APRT gene sequences with the endogenous CHO APRT locus; to determine the relative frequencies of targeted integration and gene conversion events, the effect of various-sized deletions or insertions in the target gene sequence on recombination frequencies, and whether transcribed genes are better targets for directed homologous recombination than are non- transcribed genes. Another system, employing CHO cell hybrids constructed between different, non-overlapping APRT deletion mutants, will be used to assay spontaneous or induced frequencies of interallelic mitotic recombination between endogenous APRT alleles in their normal chromosomal environment. A third system will examine the generation of homozygosity or hemizygosity for recessive mutations in cultured cells by genetic events such as mitotic recombination, chromosome nondisjunction or multilocus deletions that are undetectable in conventional CHO and V-79 mutagenesis assays. Other studies will focus on the effect of chromosomal environment on the relative susceptibility of a gene to mutation, deletion, or recombination.