TARGETED MUTATIONS OF TYROSINE KINASE GENES IN THE MOUSE

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This project will consist of the development of methods to select for the integration of retroviral DNAs into particular host genes to generate specific mutations; and to target retroviral infection to particular cell types bearing specific cell-surface proteins. In our previous work, we have demonstrated the feasibility of generating mutations by insertional inactivation of the X-linked mouse gene encoding hypoxanthine-guanine phosphoribosyl transferase (HGPRTase). We propose to extend these studies to facilitate the disruption of other more interesting target genes, and to improve the efficiency of the process. We will attempt to select for transformants due to the disruption of recessive anti-oncogenes, recently demonstrated for the retinoblastoma system. Another powerful selection is survival of cells after induction toward terminal differentiation. We will also attempt to select for insertion of retroviral genomes into genes regulated in particular ways; this procedure is analogous to the Mud-lac gene fusions used in bacteria. Lastly, we describe efforts to improve the efficiency of the targeting of retroviral DNAs to particular host genes by the incorporation into the viral genome of potential regions of homology with the target gene. In a separate subproject, we will try to target virus to particular cell types. In some of these experiments the viral env gene will be partially disrupted and substituted with the gene encoding the Staphlococcus aureus protein A. Exposure of these virus to particular monoclonal IgG preparations will coat the virus with antibody, oriented with the variable specificity-determining domain outward. The ability of these viruses to enter and grow on cells expressing the antigen recognized by the monoclonal antibody will be tested by measuring the production of progeny virus after infection.