EXPRESSION OF GENETIC VARIATION IN CULTURED CELLS

重组在产生和限制中都起着重要的作用。 活着的有机体的遗传多样性。它代表了世界上主要的 产生和表达体细胞突变的机制，并已 已被证明与多种类型的肿瘤发生有关(视网膜母细胞瘤， 等)。 矛盾的是，在正常细胞中，相同的重组途径可能 为保存基因组稳定性和提供重要的机制 DNA的修复。目前，人们对这些重组蛋白知之甚少 哺乳动物细胞的机制这个项目的长期目标是 了解细胞过程和分子机制参与的 哺乳动物体细胞可遗传变异的产生和表达 细胞。一组独特的突变细胞系和系统用于分析 中国仓鼠内源基因座上的同源重组 腺嘌呤磷酸核糖基转移酶(APRT)基因座将被用来 关于同源重组几个基本方面的问题 在哺乳动物的体细胞中。这项建议的具体目的是：(I) 研究共享序列同源性和内部序列长度的影响 靶向重组频率的序列异质性 哺乳动物细胞，使用一系列不同长度的aprt基因片段作为 靶向纠正三碱基缺失的供体序列 内源aprt基因座；(Ii)检测转录的影响 靶基因和供体序列对靶基因和供体序列频率的影响 利用靶基因在哺乳动物细胞中进行直接重复重组 替换/整合策略以获得其中 内源性aprt基因已被aprt编码序列驱动 通过异源的、可诱导的金属硫蛋白启动子，(Iii)检测 细胞周期时相对靶向同源基因频率的影响 中国仓鼠细胞内源性aprt基因的重组，通过 同步淋洗中靶向重组频率的测定 (Iv)利用基因打靶技术研究 170千碱基基因组区域的染色体内重组 在内源aprt基因的侧翼。我们希望这样的研究将 提供了对重组机制的相当深入的见解 哺乳动物的体细胞。

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.