A Transgenic System for Targeted Ablation of Reproductive and Maternal-Effectgenes

一种靶向消融生殖和母体效应基因的转基因系统

“A Transgenic System for Targeted Ablation of Reproductive and Maternal-Effect genes in Zebrafish” Maternally provided gene products regulate the earliest events of embryonic life, including formation of the oocyte that will develop into an egg, and eventually an embryo. Forward genetic screens have provided invaluable insights into the molecular regulation of embryonic development, including essential contributions of some genes whose products must be provided to the transcriptionally silent early embryo for normal embryogenesis, maternal-effect genes. However, other maternal-effect genes are not accessible due to their essential zygotic functions during embryonic development. To identify these factors it is necessary to bypass the early requirement of the gene so potential later functions of the gene can be investigated. Methods to circumvent these zygotic lethal phenotypes in the zebrafish were pioneered by Brian Ciruna, Alex Schier, and Erez Raz. Their germline replacement approach takes advantage of the early separation of somatic and germline lineages in zebrafish to generate animals with a normal somatic composition and a mutant germline through host germline ablation and transplantation (replacement) with mutant donor germ cells. This strategy allows the animal to survive to produce mutant gametes, which can be examined for reproductive and maternal-effect phenotypes. Although this approach has been applied to examine the function of specific genes, thus far, no systematic germline replacement screen of the existing large collection of zebrafish zygotic lethal mutations has been reported. We are proposing a reverse genetic system designed to identify genes whose reproductive and maternal-effect functions are masked by their essential zygotic roles in early embryogenesis. Identifying these regulators is essential to fill the large gaps in our understanding of the mechanisms and molecular pathways contributing to fertility and maternally regulated developmental processes. The goal of the studies proposed here is to develop and apply a robust and efficient genetic method to mutate the host germline and to conduct a proof of concept screen of genes known to have adult reproductive and maternal-effect phenotypes, including vasa, Kif5Ba, and bucky ball.