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

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Our research interests are focused on 1) the mapping and cloning of a cellular senescence gene on human chromosome 1 whose introduction into the human sarcoma cell line, TE85, induces senescence and on 2) clarifying normal functions of mismatch repair genes and their relationship to environmental carcinogenesis. For the first project, we plan to perform genetic complementation studies by transferring decreasing sized genetic units, including a whole chromosome 1, truncated chromosome 1, subchromosomal fragments and YAC clones from chromosome 1 to TE85. To facilitate this process, we have developed a human gene and chromosome targeting system. Using recombination-proficient chicken/human cell hybrids containing human chromosome 1, we plan to create a truncated transferrable chromosome 1 at a specific gene locus by the targeted insertion of a telomere sequence. These truncated chromosomes 1 are to be transferred into TE85 to map the senescence-inducing activity. Once the activity is mapped to region whose size is less than 1 Mb, we will isolate YAC clones from the region using our YAC cloning method which allowsone to isolate a specific region of DNA by gene targeting. Developing a minichromosome or an artificial chromosome carrying a genetic unit whose size is larger than YAC clones but smaller than a chromosome or chromosomal fragment will facilitate gene mapping as well as functional analysis of a specific gene in native form. To this goal, we plan to truncate chromosomes 1, 2, 8 and 11 at both sides of the centromere and create a minichromosome which could carry various genes and chromosomal fragments. Also, the centromere region will be isolated into YAC clones and used for constructing a YAC based artificial chromosome. We also plan to isolate DT40 cells with additional human chromosomes. For the second project, we plan to generate transferrable chromosome 2 with disrupted hMSH2 using chicken/human chromosome 2 targeting system. We plan to transfer normal and modified chromosome 2 into the hMSH2-deficient cell line, LoVo, to determine the functions of hMSH2. We also plan to generate a gene targeting vector containing a wild type hMLH1 exon 9 which is mutated in the colon cancer cell line, HCT116. We plan to replace the mutated exon 9 with a wild-type exon 9 in HCT116 by gene targeting to determine the normal functions of hMLH1.