This proposal aims at developing an applying a gene trap genetic screen method based on mobilization of transposons in zebrafish. Three fish lines will be generated. (1) The jump starter line will express transposase in the germ line cells. (2) The mutator line will contain engineered transposons, which will transpose upon being supplied with transposase activity in trans. (3) The reporter line will, in cells supplied with Gal4 from the engineered transposon, express EGFP, which will be under the control of Ga14-UAS. Genetic screen will be performed by crossing the jump starter with the mutator to generate G0. In the germ line of the G0, transposition events will take place and some of the events will lead to gene traps producing Ga14. Crossing GO with the reporter will cause the expression the EGFP in F1 embryos in patterns representing those disrupted endogenous genes. EGFP expression patterns can be visualized by fluorescence. Integration sites giving interesting EGFP patterns will be subjected to cloning, which will be a quick process. With the genes identified and mutants generated in this screen, we will greatly accelerate our understanding of the mechanisms underlying the vertebrate embryogenesis as well as human congenital diseases.
