Zebrafish (Danio rerio) is an outstanding model of vertebrate development and human disease. The field has grown enormously in recent years and made major contributions to our understanding of the genes and mechanisms underlying human developmental defects. However, two of the most important tools that made mice a premier model system, site-directed mutagenesis and targeted insertion of alterations into a specific gene, are lacking in zebrafish. The overall aim of this project, which is a collaboration between a lab with expertise in zebrafish developmental genetics and a lab with expertise in the biochemistry of recombination, is to rectify this problem by developing gene targeting technology for zebrafish. Our approach is adapted from successful experiments in Drosophila and is based on cleavage of the chosen target gene with designed enzymes called zinc finger nucleases (ZFNs). The proposed research will express ZFNs in zebrafish embryos to generate a double-strand break in a target gene, which stimulates local recombination. Non-homologous end joining introduces sequence changes that result in small mutations in the target gene, allowing targeted mutagenesis (gene knock-out) in zebrafish. ZFN-induced double-strand breaks in the target gene in the presence of engineered donor molecules leads to homologous recombination, allowing precise introduction of any desired sequence into the targeted gene (gene knock-in). This exploratory proposal uses three target sequences in two developmentally significant genes to analyze the utility of ZFN- induced mutagenesis in zebrafish. We will develop this technology with the goal of bringing the advantages of targeted gene knock-out and knock-in technology to the zebrafish research community. These novel approaches to manipulate the zebrafish genome will allow the creation of better models of a wide range of human developmental defects and diseases. [unreadable] [unreadable]