The purpose of the proposed research is to develop methods to create immune protected universal donor cloned transgenic pigs for xenotransplantation. According to the United Network of Organ Sharing (UNOS) more than 30,000 organ transplants are performed in the US alone, yet greater than 95,000 people are currently on the waiting list to receive donors organs for the treatment of end-stage organ failure. Current strategies to increase the pool of donor organs available for transplantation has only led to marginal increases that are insufficient to satisfy the growing need for donor organs and tissues. Many clinical investigators, large international pharmaceutical companies as well as several biotechnology companies have looked to develop transgenic pigs as a source of universal donor tissue. Therefore, the goal of this research plan for the Phase I STTR, is to perfect nuclear transfer with primary cell lines derived from previously cloned animals containing the human H-transferase gene and a homozygous targeted mutation of the porcine Gal-alpha1,3Gal transferase gene (GT-/-HT) into in vitro cultured pig oocytes. Primary embryonic fibroblasts and embryonic neural stem cells will be tested to determine which cell type is the best nuclear donor in in vitro nuclear transfer experiments by assessing blastocyst development (Experiment 1). Subsequently, the cell type from Experiment 1 that is most efficacious in producing blastocysts will be genetically modified with a bi-functional chimeric molecule (CD152-CD59) that inhibits both humoral and cellular immune responses to transplanted tissues. We will select transfected cell clones and analyze the cells in vitro to asses expression levels and the function of the transfected gene product. Different transfected cell clones will then be selected for follow-on nuclear transfer experiments to demonstrate blastocyst formation in vitro from the transfected cells. Demonstrating blastocyst development in vitro from cells derived from a previously cloned transgenic pig that are then genetically modified further with a molecule designed to resist immune destruction will provide the necessary data to support a Phase II STTR research proposal for developing cloned living animals that could then be used to assess survival in in vivo transplant models, which could eventually lead to clinical transplantation applications. ? ? ?