HOMOLOGOUS RECOMBINATION IN HUMAN CELL EXTRACTS

人类细胞提取物中的同源重组

• 批准号: 3290805
• 负责人: Raju S. Kucherlapati
• 金额: $ 19.95万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-06-01 至 1991-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3290805
• 关键词: DNA; affinity chromatography; bacterial virus; cell free system; cell transformation; chromosome deletion; complementary DNA; endonuclease; enzyme mechanism; gene conversion; gene expression; gene therapy; genetic library; genetic manipulation; human tissue; immunochemistry; laboratory mouse; laboratory rabbit; monoclonal antibody; plasmids; testis

The ability to modify genes in living cells by homologous recombination has significance to gene replacement therapy as well as site specific gene modification. During the past few years it has been demonstrated that mammalian somatic cells have all the enzymes necessary to mediate homologous recombination between exogenously introduced plasmids. The mechanism of this process and the actual nature of enzymes mediating the reactions are not known. As a first step towards a detailed genetic, biochemical and molecular biological understanding of mammalian honologous recombination, we have developed a cell-free system which is capable of catalyzing this reaction. The assay involves co-incubation of two non reverting, non-overlapping deletion mutants of PSV2neo, a eukaryotic-prokaryotic shuttle vector with the cell extracts and using the resulting DNA for direct analysis or transformation of recombination deficient (recA-) E-coli. The current proposal involves a detailed analysis of the cell extracts. The PSV2neo plasmids will be modified so that the two plasmids will have different restriction enzyme sites. THese restriction enzyme site polymorphisms will be used to examine the products of recombination and deduce the role of gene conversion and reciprocal recombination. The cell extracts will be tested for the presence of enzymatic activities that are required for the initiation and completion of the recombination reaction. Both biological and biochemical assays will be used. Isolation of a recombinase protein which would be involved in a DNA strand exchange reaction by fractionation methods and by antibody affinity columns is also proposed. A highly specific antibody will also be used to screen a human cDNA library in a bacteriophage expression vector to isolate the recombinase cDNA. The cDNA will be inserted into mammalian expression vectors and its ability to enhance homologous recombination will be studies. Isolation of a genomic clone corresponding to the cDNA and its characterization are also proposed.

通过同源重组修饰活细胞中基因的能力具有 位点特异性基因对基因替代治疗意义 改性 在过去几年中，已经证明， 哺乳动物体细胞具有所有必要的酶来介导 外源导入质粒之间的同源重组。 的 这一过程的机制和酶介导的实际性质， 反应是未知的。 作为详细基因分析的第一步， 哺乳动物激素的生物化学和分子生物学认识 重组，我们已经开发出一种无细胞系统， 催化这个反应。 该测定涉及两种非特异性抗体的共孵育。 PSV 2neo的回复、非重叠缺失突变体，a 真核-原核穿梭载体与细胞提取物，并使用 用于直接分析或重组转化的所得DNA recA缺陷型大肠杆菌。 目前的建议涉及一个详细的 细胞提取物的分析。 将对PSV 2neo质粒进行修饰， 这两个质粒会有不同的限制性内切酶位点 这些 限制酶位点多态性将用于检测产物 的重组和推导的作用，基因转换和互惠 重组 将检测细胞浸提液中是否存在 启动和完成所需的酶活性 重组反应。 生物学和生物化学测定都将是 采用 分离将参与DNA的重组酶蛋白 通过分级分离方法和通过抗体亲和力的链交换反应 也提出了专栏。 还将使用高度特异性抗体来 在噬菌体表达载体中筛选人cDNA文库以分离 重组酶cDNA。 将cDNA插入哺乳动物表达系统中， 载体及其增强同源重组的能力将是 问题研究 分离对应于该cDNA的基因组克隆及其表达产物， 还提出了表征。