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

我们的研究兴趣是定位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的关系。为了促进这一过程，我们利用重组熟练的鸡DT40/人类细胞杂交体开发了人类基因打靶系统。鸡DT40细胞中的任何人类基因或染色体都可以被高频率地靶向和修饰。我们开发了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，它允许人们通过基因打靶来分离DNA片段的特定区域；2)一种系统，它允许人们通过破坏基因(基因敲除)来评估特定基因的功能；3)一种系统，它允许人们通过定向插入端粒序列来在特定基因位置创建截断的可转移染色体。我们的研究项目之一是在人类1号染色体上鉴定一个新的细胞衰老基因，该基因导入人骨肉瘤细胞系TE85后会导致生长停滞。为了将基因定位到合适大小的染色体区域(小于1Mb)，我们计划通过将尺寸减小的遗传单位，包括完整染色体、截断染色体、亚染色体片段、YAC克隆和BAC克隆，转移到TE 85细胞进行遗传互补研究。为了促进这一过程，我们培育了包含可移植人类1号染色体的小鼠A9杂交细胞系，该染色体在该染色体的不同位置被截断。我们目前正在将A9细胞中的这些染色体转移到TE85，以将生长停滞活性映射到染色体1Q上40Mb的区域。为了进一步缩小衰老活性，我们计划开发一个微型染色体或人工染色体，携带一个遗传单位，其大小比粘粒克隆大，但比染色体片段小。这将有助于基因作图以及单基因的功能分析。来自人类11号染色体的微小染色体将在DT40中通过将端粒序列定向插入到该染色体的着丝粒中而产生。此外，11号染色体着丝粒区域已被分离成YAC克隆，并将用于构建基于YAC的人工染色体。为了确定人类错配修复基因hMSH2的功能，我们在DT40细胞中用修饰的hMSH2产生了可转移的人类2号染色体。修改后的2号染色体将被转移到hMSH2缺陷细胞系LoVo中，以确定hMSH2的功能。-基因打靶、同源重组、基因敲除、酵母人工染色体、端粒

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 cells can be targeted and modified at high frequency. We have developed 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 transferrable 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, induces growth arrest. To localize a gene to a clonable size of the chromosomal region (less than 1 Mb), we plan to perform genetic complementation studies by transferring decreasing sized genetic units, including whole chromosomes, truncated chromosomes, subchromosomal fragments, YAC clones and BAC clones, to TE 85 cells. To facilitate this process, we have generated mouse A9 hybrid cell lines containing a transferable human chromosome 1 truncated at various sites of this chromosome. We are currently transferring these chromosomes in A9 cells to TE85 to map the growth arrest activity to a region of 40 Mb on chromosome 1q. To further narrow down the senescence activity, we plan to develop a minichromosome or an artificial chromosome carrying a genetic unit whose size is larger than a cosmid clone but smaller than a chromosomal fragment. This will facilitate gene mapping as well as functional analysis of a single gene. The minichromosome derived from a human chromosome 11 will be generated in DT40 by targeted insertion of a telomere sequence into the centromere of this chromosome. Also, the centromere region of chromosome 11 has been isolated into YAC clones and will be used for constructing a YAC based artificial chromosome. To determine the functions of the human mismatch repair gene, hMSH2, we have generated transferrable human chromosome 2 with a modified hMSH2 in DT40 cells. Modified chromosome 2 will be transferred into the hMSH2-defective cell line, LoVo, to determine the functions of the hMSH2. - gene targeting, homologous recombination, gene knock-out, yeast arteficial chromosomes, telomere