Nuclear cloning, epigenetic reprogramming, and cellular differentiation.

The full-term development of sheep, cows, goats, pigs and mice has been achieved through the transfer of somatic cell nuclei into enucleated oocytes. Despite these successes, mammalian cloning remains an inefficient process, with a preponderance of reconstructed embryos failing at early- to mid-gestation stages of development. The small percentage of conceptuses that survive to term are characterized by a high mortality rate and frequently display grossly increased placental and birth weights. It is likely that inappropriate expression of key developmental genes may contribute to lethality of cloned embryos. One of the most interesting issues of nuclear cloning is the question of genomic reprogramming, i.e. whether successful cloning requires the resetting of epigenetic modifications which are characteristic of the adult donor nucleus. Processes such as X-inactivation and genomic imprinting are known to depend on epigenetic modifications of the genome. The classical nuclear transfer experiments with frogs have suggested that the source of the donor nucleus affects the phenotype of the clone. We have, using expression profiling, compared gene expression in clones derived from embryonic stem (ES) cells and from somatic donor cell nuclei and find substantial gene dysregulation. Our results suggest that faulty reprogramming is caused by the nuclear cloning procedure itself. In addition, the type of donor nucleus contributes to the abnormal expression pattern seen in cloned animals. One of the major unresolved issues has been whether nuclei of terminally differentiated cells can be reprogrammed by transfer into the oocyte. To address this question we have derived monoclonal mice from B and T cells and used the genetic rearrangements of the immunoglobulin and T cell receptor genes to retrospectively verify the differentiation state of the donor nucleus. Finally, we discuss our recent studies on the reprogramming of nuclei from terminally differentiated neurons and from cancer cells.