The objective of the proposed research is to develop novel approaches and/or adapt currently available techniques for genetic modification of a variety of laboratory animal species. Currently, mice are the most common species of animal used in research. Their popularity derives from their small size, low relative cost to breed and maintain, short gestation, and large litter size, as well as the vast array of commercially-available reagents and technologies. One of the greatest advances in animal model development has been the generation of techniques for producing mice that express foreign DNA (transgenic mice, knock-in mice) or have a native gene deleted (knock-out, or gene-deficient, mice). A major drawback to using mice as animal models is that they often fail to reliably mimic human development or disease. However, many of the techniques used to modify the mouse genome have been difficult to adapt to non-rodent species or are cost prohibitive to attempt in other species. Several methods have been developed to overcome these obstacles, and several recent reports have described a highly successful method for generating gene deficient rats using zinc finger nucleases (ZFNs). Briefly, ZFNs are synthetic proteins consisting of an engineered DNA- binding domain fused to an endonuclease that induce double-stranded breaks in specific DNA sequences. The DNA breaks are repaired by host cell repair pathways, with a proportion of the repairs incorporating errors resulting in genetic mutations that often translate, in effect, to deletion of the targeted gene product. The overall goal of the project described here is to use ZFN technology in the development of reliable and cost-effective methods for producing genetically modified animals that is applicable across species. Sperm- mediated gene transfer (SMGT) will be used to deliver the ZFN to the developing embryo. Using this method, freshly isolated sperm will be incubated with DNA encoding the ZFN. DNA binds to specific proteins on the surface of the sperm and is transported intracellularly. DNA-laden sperm can then be used to fertilize ova in vitro or in vivo. This technology will be compared in two species, one rodent and one non-rodent, to demonstrate proof of concept and to generate genetically modified guinea pigs and sheep that are valuable models for human diseases. The specific aims of the project are to generate 1) guinea pigs deficient in one or more genes involved in the generation of immune responses and 2) sheep deficient in one or more genes involved in embryonic development of the cardiac and respiratory systems.