In both male and female mammals, gametogenesis is a complex process that includes genetic and epigenetic programming mechanisms that prepare the gametic genomes to direct development of an ensuing embryo. When a cloned individual is produced by transfer of a somatic cell nucleus into an enucleated egg, the donor genome has NOT undergone any of the reprogramming processes unique to gametogenesis. Thus the transplanted somatic cell nucleus must respond to signals from the recipient ooplasm directing it to undergo rapid reprogramming to facilitate embryonic development. In recent studies the extent of epigenetic reprogramming in somatic cells of cloned mice has been partially analyzed, and found to be incomplete and highly variable. However, no studies have been carried out to determine the extent to which epigenetic programming is restored in germ cells of cloned mice. Furthermore, there have been no previous studies of genetic programming in any cell type in cloned mice. In this application, we propose a systematic study to compare the extent to which genetic and epigenetic parameters become properly reprogrammed in somatic and germ cells of embryos and offspring produced by cloning via the nuclear transfer (NT) method. Only a small proportion (less than 2-3%) of cloned embryos develop to term, and only a subset of these grow into fertile adults. We hypothesize that while genetic and epigenetic programming is variable and incomplete in somatic cells of cloned mice, it can be largely restored in the germline of these individuals. We further suspect that for a cloned embryo to develop normally, the donor nucleus must undergo rapid preprogramming of epigenetic mechanisms with a low frequency of mutations. Finally, we suspect that nuclei from somatic cells and germ cells differ in their initial states of genetic and epigenetic programming and hence in their ability to successfully direct embryonic development following NT. The results of these experiments will contribute to two general areas of interest. First, they will allow us to better understand how germline-specific mechanisms of genetic and epigenetic reprogramming normally function. Second, they will provide important new insight into the efficacy and safety of mammalian cloning.
