TARGETING OF HUMAN GENES AND ITS APPLICATION TO GENE MAPPING AND CLONING

人类基因靶向及其在基因图谱和克隆中的应用

• 批准号: 6106621
• 负责人: MINORU KOI
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6106621
• 关键词: DNA damage; DNA repair; chromosome deletion; gene targeting; genetic mapping; genetic markers; human genetic material tag; molecular cloning; neoplasm /cancer genetics; nucleic acid sequence; tissue /cell culture

Our research interests are focused on 1) the mapping and cloning of a cellular senescence gene on human chromosome 1 whose introduction into the human sarcoma cell line, TE85, induces senescence and on 2) clarifying normal functions of mismatch repair genes and their relationship to environmental carcinogenesis. For the first project, we plan to perform genetic complementation studies by transferring decreasing sized genetic units, including a whole chromosome 1, truncated chromosome 1, subchromosomal fragments and YAC clones from chromosome 1 to TE85. To facilitate this process, we have developed a human gene and chromosome targeting system. Using recombination-proficient chicken/human cell hybrids containing human chromosome 1, we plan to create a truncated transferrable chromosome 1 at a specific gene locus by the targeted insertion of a telomere sequence. These truncated chromosomes 1 are to be transferred into TE85 to map the senescence-inducing activity. Once the activity is mapped to region whose size is less than 1 Mb, we will isolate YAC clones from the region using our YAC cloning method which allowsone to isolate a specific region of DNA by gene targeting. Developing a minichromosome or an artificial chromosome carrying a genetic unit whose size is larger than YAC clones but smaller than a chromosome or chromosomal fragment will facilitate gene mapping as well as functional analysis of a specific gene in native form. To this goal, we plan to truncate chromosomes 1, 2, 8 and 11 at both sides of the centromere and create a minichromosome which could carry various genes and chromosomal fragments. Also, the centromere region will be isolated into YAC clones and used for constructing a YAC based artificial chromosome. We also plan to isolate DT40 cells with additional human chromosomes. For the second project, we plan to generate transferrable chromosome 2 with disrupted hMSH2 using chicken/human chromosome 2 targeting system. We plan to transfer normal and modified chromosome 2 into the hMSH2-deficient cell line, LoVo, to determine the functions of hMSH2. We also plan to generate a gene targeting vector containing a wild type hMLH1 exon 9 which is mutated in the colon cancer cell line, HCT116. We plan to replace the mutated exon 9 with a wild-type exon 9 in HCT116 by gene targeting to determine the normal functions of hMLH1.

我们的研究兴趣集中在1）映射 以及克隆人1号染色体上的细胞衰老基因 将其导入人肉瘤细胞系TE 85，诱导 衰老和2）阐明错配修复的正常功能 基因及其与环境致癌作用的关系。为 第一个项目，我们计划进行基因互补， 通过转移大小递减的遗传单位进行研究，包括 完整的1号染色体，截短的1号染色体，亚染色体 片段和YAC克隆。到 为了促进这一过程，我们开发了一种人类基因， 染色体靶向系统使用重组熟练 鸡/人细胞杂交含有人类1号染色体，我们 计划在一个特定的位置创建一个截短的可转移的1号染色体， 通过端粒序列的靶向插入，在基因位点上进行修饰。这些 将截短的1号染色体转移到TE 85中进行作图 衰老诱导活性。一旦活动映射到 区域的大小小于1 Mb，我们将分离YAC克隆 使用我们的YAC克隆方法， 通过基因靶向分离DNA特定区域。开发一 微型染色体或携带遗传基因的人工染色体 一个单位的大小大于YAC克隆，但小于 染色体或染色体片段将有助于基因定位 以及天然形式的特定基因的功能分析。到 为了实现这一目标，我们计划将1、2、8和11号染色体分别截短， 着丝粒的两侧，并创建一个微型染色体， 携带各种基因和染色体片段。还 将着丝粒区域分离到YAC克隆中，并用于 构建基于YAC的人工染色体。我们还计划 分离具有额外人类染色体的DT 40细胞。为 第二个项目，我们计划产生可转移的2号染色体 使用鸡/人2号染色体， 瞄准系统我们计划把普通的和改装的 将2号染色体导入hMSH2缺陷细胞系LoVo， 确定hMSH2的功能。我们还计划制造一种基因 含有野生型hMLH1外显子9的靶向载体， 在结肠癌细胞系HCT116中突变。我们计划更换 HCT 116中突变的外显子9与野生型外显子9通过基因 以确定hMLH1的正常功能。