International Mammalian Genome Society

The 14th International Mouse Genome Conference (2000)

D8. Exploiting Bacteriophage Genetics for High Throughput Functional Genomics

D. E. Rancourt, K. Woltjen, M.W.T. Unger, G. Bain, T. Tsuzuki*
Department of Biochemistry and Molecular Biology, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta, Canada T2N 4N1 and
*Department of Medical Biophysics and Radiation Biology, Graduate School of Medical Sciences, Kyushu University, 1-1, Maidashi 3-chome, Higashi-ku, Fukuoka, 812-8582, Japan.

Targeted mutagenesis is an extremely useful tool in molecular medicine, allowing the generation of customized mutant mice and animal models of disease. With the predicted identification of 100,000 genes in the mammalian genome, targeted mutagenesis is expected to play an ever-increasing role in determining gene function, and in validating targets for rational drug design. In anticipation of the demand for efficient functional genomics, we have begun to develop novel technologies that shorten the targeted mutagenesis timeline. Using bacteriophage as a system for gene targeting, we demonstrate that targeting vectors can be rapidly assembled using double-crossover phage-plasmid recombination. In addition to directing insertion cassettes into particular exons, novel transplacement strategies facilitate the transfer of custom mutations to phage vectors. Using bacteriophage genetics, we demonstrate that specific targeting vectors can be genetically isolated from a TK targeting vector library. In combination, these systems allow for phage TVs to be rapidly pulled from a library and simultaneously modified by phage-plasmid recombination. Thus, TVs can be easily and quickly generated in bacteriophage without plasmid subcloning and with little genomic sequence or restriction site information. (Supported by the Alberta Cancer Board, Alberta Heritage Foundation for Medical Research, Natural Sciences and Engineering Council of Canada and Ministry of Education, Science, Sports and Culture of Japan).

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