The zebrafish has characteristics that make it an ideal model organism for studying genetic determinants that participate in development and disease. However, one major limitation to its widespread use is the absence of a technology that allows direct genomic manipulation of a desired target gene either to inactivate or alter its function. Recent studies in human cell lines, insects and plants indicate that site-specific mutations can be introduced using zinc finger nucleases (ZFNs). Using a combination of rational design and selection, we have successfully engineered ZFNs that promote targeted gene disruption in zebrafish that are transmissible through the germline. Using this technology it is now possible to generate targeted mutations throughout the zebrafish genome. In this grant proposal, we propose to optimize and extend the use of ZFNs in zebrafish. A major goal of the proposed studies will be to enable individual researchers to generate knockout or knock-in zebrafish lines in their own laboratories with modest technical effort. We will streamline the creation of site- specific zinc finger proteins the targeting component of ZFNs to simplify the process of making gene knockouts. We will further utilize and optimize ZFN technology for targeted homologous recombination with a DNA donor supplied in trans allowing targeted gene knock-ins. We will demonstrate the utility of this technology by making conditional alleles of genes involved in heart regeneration to allow the study of knockouts of required genes in the adult. We will also generate an allelic series of mutations in the flt4 receptor tyrosine kinase to better understand its role at multiple steps in vascular development. By providing an accessible technological platform for genetic manipulation to the zebrafish community, this study will ultimately enable the creation of a variety of gene knockouts or knock-ins that can be used in conjunction with powerful forward and reverse genetic screens in the zebrafish to study human disease models. Thus, the development and use of ZFN technology in the zebrafish will have far- reaching significance in virtually all areas of biomedical research. PUBLIC HEALTH RELEVANCE: We are developing technology to allow the genome of the zebrafish to be engineered. This technology will allow researchers to construct models of human disease in this animal to understand its origin and develop better treatments. We will apply this technology in zebrafish to better understand the processes involved in heart regeneration and blood vessel formation.