EXPRESSION OF GENETIC VARIATION IN CULTURED CELLS

培养细胞中遗传变异的表达

• 批准号: 3168302
• 负责人: GERALD M ADAIR
• 金额: $ 18.6万
• 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 1995-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3168302
• 关键词: adenine phosphoribosyltransferase; autosomal recessive trait; biochemical evolution; 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; genetic transcription; hamsters; heterozygote; homozygote; hybrid cells; metallothionein; molecular pathology; mutant; nucleic acid sequence; plasmids; tissue /cell culture; transposon /insertion element; trisomy

Recombination plays an important role in both the generation and limitation of genetic diversity in living organisms. It represents one of the major mechanisms for the generation and expression of somatic mutations, and has been shown to be involved in many types of tumorigenesis (retinoblastoma, etc.). Paradoxically, in normal cells, the same recombinational pathways may provide important mechanisms for the preservation of genomic stability and the repair of DNA. At present, little is known about these recombinational mechanisms in mammalian cells The long-term goal of this project is to understand the cellular processes and molecular mechanisms involved in the generation and expression of heritable variation in mammalian somatic cells. A unique set of mutant cell lines and systems for the analysis of homologous recombination at an endogenous gene locus-the Chinese hamster adenine phosphoribosyltransferase (APRT) locus, will be used to address questions regarding several fundamental aspects of homologous recombination in mammalian somatic cells. The specific aims of this proposal are: (i) to investigate the effects of length of shared sequence homology and internal sequence heterologies on the frequency of targeted recombination in mammalian cells, using a series of different length APRT gene fragments as donor sequences for targeted correction of a three-basepair deletion at the endogenous APRT locus; (ii) to examine the effects of transcription of target gene and donor sequences on the frequency of targeted and direct-repeat recombination in mammalian cells, by employing targeted gene replacement/ integration strategies to obtain cell lines in which the endogenous APRT gene has been replaced with an APRT coding sequence driven by a heterologous, inducible metallothionein promoter, (iii) to examine the effects of cell cycle phase on the frequency of targeted homologous recombination at the endogenous APRT locus in Chinese hamster cells, by assaying targeted recombination frequencies in elutriationsynchronized populations; and (iv) to utilize gene targeting techniques to study intrachromosomal recombination in a 170 kilobasepair genomic region flanking the endogenous APRT locus. It is our hope that such studies will provide considerable insight into the mechanisms of recombination in mammalian somatic cells.

无论是在生成还是限制过程中， 生物体的遗传多样性。它代表了一个主要的 体细胞突变的产生和表达的机制， 已显示参与许多类型的肿瘤发生（视网膜母细胞瘤， 等）。 奇怪的是，在正常细胞中，相同的重组途径可能 为基因组稳定性的保存提供重要的机制， DNA的修复。目前，人们对这些重组 该项目的长期目标是， 了解参与的细胞过程和分子机制， 哺乳动物体细胞遗传变异的产生和表达 细胞一套独特的突变细胞系和系统，用于分析 中国仓鼠内源基因座的同源重组 腺嘌呤磷酸核糖转移酶（APRT）基因座，将用于解决 关于同源重组的几个基本方面的问题 在哺乳动物体细胞中。这项建议的具体目标是： 研究共享序列同源性长度和内部 序列异质性对靶向重组频率的影响 哺乳动物细胞，使用一系列不同长度的APRT基因片段作为 供体序列，用于靶向校正在所述位点处的三个碱基对缺失。 内源性APRT基因座;（ii）检查转录的影响， 靶基因和供体序列对靶向和 在哺乳动物细胞中直接重复重组，通过使用靶基因 替代/整合策略以获得细胞系，其中 内源性APRT基因已被驱动的APRT编码序列取代 通过异源的、可诱导的金属硫蛋白启动子，（iii）检查 细胞周期时相对靶向同源 在中国仓鼠细胞中内源性APRT基因座的重组， 分析淘洗中的靶向重组频率 （四）利用基因打靶技术研究 在170个内切酶对基因组区域中染色体内重组 位于内源性APRT基因座的侧翼。我们希望这些研究将 提供相当深入的重组机制， 哺乳动物体细胞