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: 5202163
• 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/5202163
• 关键词: DNA repair; animal tissue; ataxia telangiectasia; cell senescence; chromosome deletion; gene targeting; genetic mapping; genetic markers; genetic recombination; human genetic material tag; molecular cloning; neoplasm /cancer genetics; nucleic acid sequence; southern blotting; telomere; tissue /cell culture; transfection

This project is to establish methods which allow one to target human genes in chicken B cell line,DT40, which has high homologous recombination activity. It has been reported that the exoginous DNA sequences integrate into homologous sequences in chicken genome by transfection of DT40 cells. If one can transfer a single copy of a human chromosome into DT40 and establish the monochromosomal hybrids, one might be able to target human genes in DT40. We transferred human chromosomes 1,2,3,11 and X from mouse A9 hybrids containing each of these chromosomes to DT40. DT40 cell lines containing human chromosomes 1,2,3 and 11 were successfully constructed. To test whether human genes can be targeted in DT40 hybrids by exoginous sequence, two gene loci, D11S16 locus on chromosome 11p13 and HRAS on chromosome 11p15 were chosen. Targeting constructs containing the D11S16 and HRAS sequences and zeomycin- resistant gene (mamalian selectable marker) were made and transfected to DT40 cells containing a human chromosome 11. Ten zeomycine-resistant clones from each transfection experiment were isolated. Southern blot analyses showed 60-70% of these clones were targeted, supporting the high frequency of a targeted integration of exoginous sequence. 1)We will generate DT40 hybrids containing human chromosomes 4 to 22. 2)We will generate chromosomal fragments from chromosome 1,3 and 11 for cellular senescence gene mapping, 3)We will knock-out the specific genes including mismatch repair genes hMSH2 on chromosome 2, hMLH1 on chromosome 3, and Ataxia Telangiectasia gene on chromosome 11 to determine the biological functions of these genes, 3) we will introduce a yeast selection marker to the specific gene loci to clone the specific regions into YACs, 4) we will introduce telomere sequence into specific gene loci to generate new chromosome with known deletion. These efforts will contribute to the mapping and cloning of novel cancer related genes.

该项目旨在建立一种方法， 与鸡B细胞系DT 40中的同源性较高， 重组活性据报道，外源DNA 序列整合到鸡基因组中的同源序列中， 转染DT 40细胞。如果一个人能把一个复制的人类 染色体插入DT 40并建立单染色体杂交种， 能够靶向DT 40中的人类基因。我们将人类染色体 1、2、3、11和X来自含有这些染色体中的每一个的小鼠A9杂种 DT 40对含有人1、2、3和11号染色体的DT 40细胞系进行了体外培养， 成功构建为了测试人类基因是否可以靶向 DT 40杂种通过外源基因序列，两个基因位点，D11 S16位点上 选择染色体11 p13和染色体11 p15上的HRAS。靶向 含有D11 S16和HRAS序列和zeomycin的构建体， 制备抗性基因（哺乳动物选择标记）并转染， DT 40细胞含有人类11号染色体。10个耐博莱霉素的 分离来自每个转染实验的克隆。Southern印迹 分析显示，这些克隆中有60-70%是靶向的，支持高 外源序列的靶向整合频率。（1）我们将 产生含有人类染色体4至22的DT 40杂交体。（2）我们将 从染色体1、3和11产生染色体片段， 衰老基因定位，3）我们将敲除特定的基因，包括 错配修复基因hMSH 2位于2号染色体，hMLH 1位于3号染色体， 共济失调毛细血管扩张症11号染色体基因的生物学确定 这些基因的功能，3）我们将引入酵母选择标记 将特异性区域克隆到YACs中，4）我们 将端粒序列引入特定的基因位点， 已知缺失的染色体。这些努力将有助于 新的癌症相关基因的定位和克隆。