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

这个项目是建立方法，允许一个人以人类为目标 同源性较高的鸡B细胞系DT40中的基因 重组活动。据报道，外源DNA 序列整合为鸡基因组中的同源序列 DT40细胞的转染法。如果一个人能把人类的一个拷贝 将染色体导入DT40并建立单染色体杂交种，可能会 能够在DT40中定位人类基因。我们转移了人类的染色体 1，2，3，11和X来自含有这些染色体的小鼠A9杂交 转到DT40。含有人1、2、3和11号染色体的DT40细胞系 已成功构建。为了测试人类基因是否可以被靶向 DT40杂交种由外源序列、两个基因座、D11S16基因座组成 选择染色体11p13和染色体11p15上的HRAS。瞄准 含有D11S16和HRAS序列和玉米素的构建体- 制作了抗性基因(哺乳动物可选择标记)，并将其导入 含有一条人染色体的DT40细胞11.10个对玉米星耐药的细胞 分离每一次转染实验中的克隆。Southern印迹 分析显示，这些克隆中有60%-70%是有针对性的，支持最高 有针对性地整合外源序列的频率。1)我们会 产生包含人类4至22号染色体的DT40杂交种。2)我们会 从1号、3号和11号染色体生成细胞染色体片段 衰老基因定位，3)我们将敲除特定基因，包括 错配修复基因hMSH2位于2号染色体，hMLH1位于3号染色体，以及 共济失调毛细血管扩张症基因在11号染色体上的生物学测定 这些基因的功能，3)我们将引入一个酵母选择标记 针对特定的基因座，将特定区域克隆到YACs中，4)我们 将端粒序列引入特定的基因位点以产生新的 已知缺失的染色体。这些努力将有助于 肿瘤相关新基因的定位和克隆。

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.