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，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，被认为会导致生长停滞。为了将基因定位到合适大小的染色体区域，我们通过随机插入端粒序列，在小鼠A9杂交细胞中产生了在该染色体的不同位置包含截短1号染色体的细胞。令人惊讶的是，将完整的1号染色体和任何被截短的1号染色体转移到TE85中并没有诱导衰老，这表明1号染色体不携带衰老诱导基因。我们目前正在将包括1、2、3和6号染色体在内的其他人类染色体转移到TE85中，以鉴定对TE85细胞具有衰老活性的人类染色体。我们正在构建一个微型微体，它可以携带BAC或YAC克隆的单个副本，以便于基因定位和单个基因的功能分析。在DT40细胞中，通过将端粒序列定向插入到该染色体着丝粒附近的位置，将从11号染色体产生一个微小染色体。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缺陷的人结肠细胞系HCT116中。

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.