Genetic manipulation of animals to produce animal models of disease is critical to the study of biological aspects of human disease, and is necessary for the development of therapeutic intervention in disease and an understanding of the natural history of many types of human ailment. The discovery of pluripotent embryonic stem (ES) cells from normal embryos was an important advance which allowed, for the first time, the development of mutations in genes of choice. By combining homologous recombination with the ability of these cells to form germline mosaic chimeras, it has been possible to make null mutations in genes of choice and thereby create important animal models for the analysis of the functions of genes in a range of biological process and human disease. Although ES cells have been isolated from blastocysts of several mammalian species, developing targeted mutations has only worked in the mouse to date. There are many possible reasons for this, but the importance of non-mouse species in genetic research, particularly the rat, is undeniable. Accomplishing targeted mutations in the rat has proven to be problematic, for while isolating ES cells is possible, the cells are not capable of generating germline mosaic chimeras. Even though ES cells from the rat blastocyst can be cultured and display markers of early embryonic stages, they cannot yet be used to generate genetic animal models. Recently, nuclear transfer has been shown to produce viable offspring from isolated blastomeres of several mammalian species. Cultured cells including those isolated from adult sheep have been proven to develop into viable offspring when fused to enucleated eggs. The applicants have developed methods for the stable isolation of early embryonic cells from the rat, suitable for in vitro genetic manipulation. The applicants are proposing to use these cells for nuclear transfer into recipient rat eggs. The reconstructed eggs will have the genetic information of the cultured cells, and when transferred to the reproductive tract of rat surrogate mothers will develop into offspring of the donor nuclear type, and bearing changes induced in the cultured cells. The applicants propose using these cells as a tool to take advantage of rapidly expanding rat genome information in order to establish novel animal models of human disease in the rat. The extensive data in rat make it the experimental model of choice for many carcinogenesis studies, cardiovascular studies, and neurobehavioral studies.
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