9317469 Driever The organisms best suited to study fundamental principles of vertebrate development should allow the application of large numbers of different experimental approaches: classical genetic analysis, experimental embryology, and molecular genetics, especially the generation of transgenic animals. Zebrafish (Brachydanio rerio), a small tropical freshwater teleost, is most amenable to the former two approaches, but methods for controlled and efficient generation of germline transgenic zebrafish have not been developed. The objective of the proposed research is to optimize methods for the infection of zebrafish germline cells with retroviruses which are pseudotyped with the vesicular stomatitis virus (VSV) G glycoprotein. Preliminary experiments have demonstrated that the VSV-G protein broadens the host range of murine leukemia virus derived vectors to zebrafish cells and other non-mammalian hosts. These methods will be applied to perform insertional mutagenesis screens in zebrafish to identify novel genes that are important for pattern formation and organogenesis in the embryo. Specific aim 1 is to optimize conditions for infection of zebrafish germline cells with VSV-G pseudotyped murine leukemia virus-derived vectors and to study germline transmission of integrated DNA. Specific aim 2 is to construct viral vectors for a) cell marking experiments to allow high resolution lineage analysis in the developing embryo, b) over expression of known proteins to study their function in development, and c) gene transfer of cloned zebrafish genes back into zebrafish hosts mutant for developmental regulator genes to allow tests for complementation. Specific aim 3 is to construct promoter trap and enhancer trap retroviral vectors and to perform initial screens for expression patterns that might reveal integration of the virus into developmental control genes. Retroviral vectors are superior to other means of insertional mutagenesis because of the defined s tructure of the integrated DNA, and the high efficiency at which the vector is delivered into the host cells. Because the VSV-G pseudotyped vectors infect a broad range of host cells including fish, Xenopus laevis, and insect cells, the methods and vectors developed during this project can also be applied to other non-mammalian developmental systems. ***
