Specific alterations in single genes in an otherwise normal animal is feasible by the utilization of the process of homologous recombination in pluripotent/totipotent embryonic cells. The animals generated, having fully characterized single gene changes, have the potential of being very valuable (i) for analyzing the relationship between specific genetic modification and the development of phenotypic changes associated with transplant rejection and (ii) for the development and testing of new methods of immune suppression following organ transplant. Unfortunately, to date only in the mouse species has this type of modification been possible due to the unavailability of embryonic carrier cell in other species. Previous work on the isolation of porcine ES cells has demonstrated the ability to identify and isolate ES-like cells from preimplantation porcine embryos, but it has not been possible to maintain the isolated ES-like cells for prolonged periods of time or to demonstrate their ability to contribute to the formation of ES-blastocyst chimeras. In mice, however, it is now possible to isolate pluripotent cell lines from primordial germ cells (PGCs). In preliminary experiments we have been able to isolate and genetically transform cultured porcine PGCs while retaining the typical morphology and alkaline phosphatase activity of undifferentiated pluripotential embryonic cells. We have also been able to demonstrate that transgenic porcine PGC-derived colonies have the ability to participate in chimera formation when introduced into a host blastocyst. We intend to extend these observations by proposing the following specific aims: 1) Optimization of conditions for the detection of transgenic porcine PGC- derived cells; 2) Increase the efficiency of isolation, long term maintenance, and cloning efficiency of primordial germ cell-derived cell lines; 3) The characterization of the developmental competence of transgenic PGC-derived cells; 4) Demonstrate the ability of PGC-derived cells to undergo homologous recombination. The successful completion of this project will result would demonstrate the feasibility of introducing genetic modifications in swine by homologous recombination. This will have broad and important implications in multiple areas of biomedical research.
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