Cloning by somatic cell nuclear transfer offers exciting new possibilities for basic research in embryology, development of stem cell therapies, and important agricultural applications. Cloning remains highly inefficient, and recent studies indicate that nuclear reprogramming may be defective in cloned embryos. However, no detailed study of reprogramming has been undertaken, and little information has been obtained about the phenotypic effects of incomplete reprogramming in the early embryo. We have found that early cloned mouse embryos differ substantially from normal fertilized embryos with respect to culture medium preference and DNA methyltransferase expression, exhibit enhanced glucose uptake relative to control embryos, and are deficient in post-transcriptional gene regulation. Some of these differences can be seen even before the first cleavage division, indicating an immediate effect of the donor nucleus on cloned embryo properties. We hypothesize that continued expression of the somatic cell genome after transfer, combined with the translation of maternal mRNAs present in the oocyte, generates a gene expression repertoire in the cloned embryo intermediate between that of a somatic cell and that of an embryo. This altered gene expression pattern may cause cloned embryos to differ markedly from normal embryos with regard to basic physiological and metabolic parameters. This may lead to defects in such basic functions as internal pH regulation, osmoregulation, homeostasis, and ATP production. We also hypothesize that as a result of this aberrant metabolic or physiological state, typical mouse embryo culture environments are grossly sub-optimal for the cloned embryo, and as a result the cloned embryo exists in a state of poor health that is not conducive to efficient nuclear reprogramming, leading to the low efficiency of overall success. Indeed, we find strikingly different culture requirements manifested by cloned embryos. Last, we hypothesize that different somatic cell types may differ in compatibility with the cloning process due to differences in initial donor cell state. To test these hypotheses, we will pursue three complementary and essential Aims. First, we will determine to what degree the transferred somatic cell nucleus continues to express its pre-programmed repertoire of genes. Second, we will determine to what degree these nuclei can direct embryo-specific gene expression patterns, including appropriate posttranscriptional recruitment of matemal mRNA. Third, we will determine to what degree specific metabolic and physiological aspects of cell function are altered in cloned embryos. Fulfillment of these Aims will provide novel information about basic regulatory and homeostatic mechanisms of normal embryos, and will also a basis for further studies examining the molecular basis of reprogramming, and for improving cloning success. ? ?
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