Work with other systems utilizing pluripotent embryonal stem (ES) cell cultures for the formation of chimeric animals has succeeded in creating organisms in which foreign genes are expressed, as well as organisms in which endogenous genes have been altered. Dr. Barnes is interested in developing Zebra fish ES cell cultures for derivation of chimeric transgenic animals. He developed serum-free culture techniques allowing derivation of karyotypically normal late-stage mouse embryo cells that could not be derived in conventional serum-containing medium. Applying this approach to fish embryo cells, he found a mitogenic activity in trout embryo extract that was essential for the cultures and adapted transfection techniques to achieve expression of exogenous genes in fish cell cultures. He proposes to: (1) use the mitogenic activity in trout embryos to derive ES cell cultures and characterize the cells through transfection of exogenous genes, injection into developing embryos, and evaluation of contribution to transgenic chimeras; (2) apply this approach to haploid embryos to derive haploid ES cell cultures allowing alteration of endogenous genes.