Many basic developmental and physiological mechanisms are highly conserved in all vertebrates, from human to zebrafish. This exploratory grant will test a novel strategy to assign functions to genes defined in current sequencing projects by taking advantage of the strengths of the zebrafish model system for functional genomics. The goal is to assess a unique approach to disrupt and tag essential genes at high-throughput and low cost. The proposed technology involves rapid gene disruption by retroviral insertions and subsequent mapping and identification of mutated genes by sequence database searches. The following protocol will be tested: Females carrying multiple independent retroviral insertions in the germline will be generated. These females will be used to produce gynogenetic diploid embryos, i.e. embryos that carry only the chromosome set of their mothers. Thus mutant phenotypes become apparent in a one-generation screen, and a large number of mutants can be efficiently identified. This one-generation screen will overcome the inefficiency of current insertional mutagenesis protocols, but the approach necessitates the identification of mutations apparent in small number of progeny. To circumvent this limitation, a large number of progeny will be analyzed. Subsequently, the genomic region flanking disruptive inserts is sequenced, compared to sequence databases, and positionally assigned to the zebrafish genetic map. Mutations are recovered in the next generation by raising the progeny of females that harbor mutations. It is estimated that this approach will allow one person to tag 100-200 genes/year at low cost. The proposed study will test the feasibility and robustness of this approach (proof of principle), and develop it to a stage where it can be applied widely and efficiently at a genomic scale.
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