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: 2574330
• 负责人: M 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/2574330
• 关键词: DNA damage; DNA repair; cell cycle; chromosome deletion; gene targeting; genetic mapping; genetic markers; human genetic material tag; loss of heterozygosity; molecular cloning; neoplasm /cancer genetics; nucleic acid sequence; telomerase; tissue /cell culture; transfection; tumor suppressor genes

Our efforts focused on mapping novel tumor suppressor genes(TSG) to human chromosomes 1,3,11, and X. We also studied the functions of mismatch repair genes. To map and clone functional TSGs, normal human chromosomes were transferred into several tumor cell lines. Introduction of a chromosome 1 into an endometrial carcinoma cell line and that of a chromosome 3 into a colorectal cancer cell line induced growth arrest in vitro and downregulation of telomerase activity. Introduction of a chromosome 11 into a bladder carcinoma cell line induced growth arrest without down-regulation of telomerase. We examined loss of heter- ozygosity(LOH) on the X chromosome of human ovarian carcinoma and found specific LOH at the Xq25-26.1 region, suggesting the presence of a TSG in this region. The loss was preferentially on an inactive X chromosome. To facilitate mapping and cloning of these TSGs, we developed a human gene targeting system using human chromosomes in a chicken cell line with a high frequency of homologous recombination. By inserting a functional telomere sequences into a specific gene loci of chromosomes in the chicken cells, we will generate transferable chromosomal fragments from chromosomes 1,3, and 11. These fragments containing a defined region of a chromosome will be transferred to each of the tumor cell lines to map growth arrest activities. By transferring human chromosomes 2 and 3 into mismatch repair (MMR) deficient colorectal tumor cell lines, we demonstrated that MMR defects and its associated phenotypes, including microsatellite instability and tolerance to alkylating agents are recessive. We also demonstrated that 1) MMR may be connected to the G2 cell cycle check point upon recognition of DNA damage; 2) MMR proteins may be involved in transcription-coupled excision repair pathway; 3) MMR system is involved in resistance to cisplatin. To demonstrate that the MMR genes are responsible for these phenotypes, we will generate chromosome(s) with disrupted MMR genes and transfer them to MMR-deficient cells.

我们的工作集中在定位新的肿瘤抑制基因（TSG）， 1号，3号，11号和X号染色体我们还研究了失配的作用 修复基因为了定位和克隆功能性TSGs， 转移到几种肿瘤细胞系中。引入 将1号染色体导入子宫内膜癌细胞系， 3号染色体导入大肠癌细胞系诱导生长停滞， 并下调端粒酶活性。引入 11号染色体导入膀胱癌细胞系诱导生长停滞 而不下调端粒酶。我们检查了他的心率- 在卵巢癌X染色体上发现了一个杂合性（洛）， 在Xq 25 -26.1区域存在特异性洛缺失，提示存在TSG 在这个地区。亏损 优先在非活性X上 染色体为了便于这些TSG的定位和克隆，我们开发了 在鸡细胞中使用人类染色体的人类基因靶向系统 同源重组频率很高。通过插入 将功能性端粒序列插入染色体的特定基因位点 在鸡细胞中，我们将产生可转移的染色体片段 分别来自1号3号和11号染色体这些片段包含一个确定的区域 将染色体转移到每个肿瘤细胞系中， 地图生长停滞活动。通过转移人类2号和3号染色体 在错配修复（MMR）缺陷结肠直肠肿瘤细胞系中， 证明了MMR缺陷及其相关表型，包括 微卫星不稳定性和对烷化剂的耐受性是 隐性的 我们还证明了1）MMR可能与G2 识别DNA损伤后的细胞周期检查点; 2）MMR蛋白 可能参与转录偶联切除修复途径; 3）MMR 系统参与顺铂耐药。以证明 MMR基因负责这些表型，我们将产生 染色体与破坏的MMR基因，并将它们转移到MMR缺陷 细胞