| Co-Investigator(Kenkyū-buntansha) |
ROBERTSON Ge オタワ大学, 医学部, 準教授
NAKABEPPU Yusaku Kyushu University, Medical Institute of Bioregulation Dept.of Biochemistry, Prof, 生体防御医学研究所, 教授 (30180350)
HAYAKAWA Hiroshi Kyushu University, Faculty of Medicine Dept.of Biochemistry, Research Associate, 医学部, 助手 (70150422)
RANCOURT Susan L. University of Calgary, Faculty of Medicine Dept.of Medical Biochemistry, Researc, 医学部, 助手
RNCOURT Derr カルガリー大学, 医学部, 準教授
RANCOURT Derrick E. University of Calgary, Faculty of Medicine Dept.of Medical Biochemistry, Associa
ROBERTSON George S. University of Ottawa, Faculty of Medicine Dept.of Pharmacology, Associate Profes
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| Research Abstract |
Targeted mutagenesis is an extremely useful experimental approach in molecular medicine, allowing the generation of specialized animals that are mutant for any gene of interest. Currently, the rate determining step in any gene targeting experiment is the construction of the targeting vector (TV). In order to streamline gene targeting methods and avoid problems encountered with plasmid TVs, we describe the direct application of phage in targeted mutagenesis. The recombination-proficient phage vector, 2TK,permits the generation of TVsby conventional restrictin-ligation or recombination-mediated methods. The resulting TV DNA can then be cleaved with restriction endonucleases to release the bacteriophage arms and can subsequently be electroporated directly into ES cells to yield gene targets. We demonstrate that in vivo phage-plasmid recombination can be used to introduce neo and lacZ-neo mutations into precise positions within a 2TK subclone via double-crossover recombination. We describe two methods for eliminating single-crossover recombinants : spi selection and size restriction ; both which result in phage TVs bearing double-crossover insertions. Thus TVs can be easily and quickly generated in bacteriophage without plasmid subcloning and with little genomic sequence or restriction site information.
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