EXPRESSION OF GENETIC VARIATION IN CULTURED CELLS

培养细胞中遗传变异的表达

• 批准号: 3168301
• 负责人: GERALD M ADAIR
• 金额: $ 14.62万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-07-01 至 1990-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3168301
• 关键词: adenine phosphoribosyltransferase; autosomal recessive trait; cell growth regulation; cell transformation; chromosome disorders; cytogenetics; gene conversion; gene expression; gene frequency; gene mutation; genetic manipulation; genetic mapping; genetic markers; genetic promoter element; genetic recombination; hamsters; heterozygote; homozygote; hybrid cells; molecular pathology; mutant; plasmids; tissue /cell culture; trisomy

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.

这项研究的目的是确定在多大程度上， 外突变事件，如有丝分裂重组，基因 转换、基因失活、多位点缺失和染色体 不分离或染色体重排，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， 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 参与生成的细胞过程和机制， 哺乳动物体细胞中遗传变异的表达。 一 系统，以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。 该系统将 用于研究转染或 用内源性CHO APRT显微注射APRT基因序列 基因座;以确定靶向的相对频率 整合和基因转换事件，各种大小的影响， 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， 转录基因 另一个系统，使用CHO细胞杂交体 在不同的非重叠APRT缺失之间构建 突变体，将用于测定自发或诱导频率 内源性APRT之间的等位基因间有丝分裂重组 在正常的染色体环境中。 第三系统 将检查纯合子或半合子的产生， 在培养细胞中的隐性突变， 有丝分裂重组、染色体不分离或多位点 在常规CHO和V-79中检测不到的缺失 诱变测定。 其他研究将集中在影响 染色体环境对基因相对易感性的影响 突变、缺失或重组。