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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

基本信息

批准号：
6432270
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
项目类别：
财政年份：
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/6432270
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

项目摘要

Our research interests are to map and isolate novel cancer-causing genes, to clarify their normal functions and their relationship to environmental carcinogenesis. To facilitate this process, we have developed human gene targeting system using recombination proficient chicken DT40/human cell hybrids. Any human gene or chromosome in chicken DT40 cell can be targeted and modified at high frequency. We have been developing the following three applications of this system; 1) a novel yeast artificial chromosome (YAC) cloning method which allows one to isolate a specific region of DNA segment by gene targeting; 2) a system which allows one to assess the functions of a specific gene by disruption of the gene (gene knock-out); 3) a system which allows one to create a truncated transferable chromosome at specific gene locus by targeted insertion of a telomere sequence. One of our research projects has been directed toward identifying a novel cellular senescence gene on human chromosome 1 whose introduction into the human osteosarcoma cell line, TE85, is supposed to induce growth arrest. To localize a gene to a clonable size of the chromosomal region, we generated mouse A9 hybrid cells containing a truncated chromosome 1 at various sites of this chromosome by a random insertion of a telomere sequence. Surprisingly, transfers of an intact chromosome 1 as well as any of the truncated chromosomes 1 into TE85 did not induce senescence, suggesting that chromosome 1 does not carry a senescence-inducing gene. We are currently transferring other human chromosomes including chromosomes 1, 2, 3, and 6 into TE85 to identify the human chromosome that carries senescence activity for TE85 cells. We are constructing a minicromosome, which can carry a single copy of BAC or YAC clone, to facilitate gene mapping as well as functional analysis of a single gene. A minichromosome will be generated from chromosome 11 in DT40 cells by targeted insertion of a telomere sequence into the loci close to a centromere of this chromosome. To determine the functions of the human mismatch repair gene, hMSH3, we have transferred a human chromosome 5 carrying a wild type hMSH3 into the hMSH3-defective human colon cell line, HCT116.

我们的研究兴趣是绘制和分离新的致癌基因，阐明它们的正常功能及其与环境致癌作用的关系。为了促进这一过程，我们已经开发了人类基因打靶系统使用重组熟练鸡DT 40/人细胞杂交。鸡DT 40细胞中的任何人类基因或染色体都可以被高频率靶向和修饰。我们一直在开发该系统的以下三个应用：1）一种新的酵母人工染色体（YAC）克隆方法，该方法允许通过基因打靶分离DNA片段的特定区域; 2）一种系统，该系统允许通过破坏基因来评估特定基因的功能（基因敲除）; 3）允许通过靶向插入端粒序列在特定基因座产生截短的可转移染色体的系统。我们的一个研究项目是鉴定一种新的细胞衰老基因，该基因位于人类1号染色体上，被认为可以诱导人骨肉瘤细胞系TE 85的生长停滞。为了将基因定位到染色体区域的可克隆大小，我们通过随机插入端粒序列产生了在该染色体的各个位点含有截短的染色体1的小鼠A9杂交细胞。令人惊讶的是，将完整的1号染色体以及任何截短的1号染色体转移到TE 85中不会诱导衰老，这表明1号染色体不携带衰老诱导基因。我们目前正在将其他人类染色体，包括1号、2号、3号和6号染色体转移到TE 85中，以鉴定携带TE 85细胞衰老活性的人类染色体。我们正在构建一个微型微粒体，它可以携带BAC或YAC克隆的单拷贝，以便于基因定位以及单个基因的功能分析。通过将端粒序列靶向插入到靠近该染色体的着丝粒的基因座中，将从DT 40细胞中的染色体11产生微型染色体。为了确定人类错配修复基因hMSH3的功能，我们将携带野生型hMSH3的人类5号染色体转移到hMSH3缺陷的人类结肠细胞系HCT 116中。