Epigenetic reprogramming of imprinted genes in embryonic stem cells, fertilized and cloned embryos

Given the fact that embryonic stem (ES) cells, which are derived from the inner cell mass of the blastocyst, are dissimilar to embryonic fibroblast cells in the level of DNA methylation and the enzymatic activity of methyltransferases, the general objective of my thesis was to characterize the expression pattern of a range of imprinted genes in mouse ES cells, fertilized and cloned embryos, all of which have been pretreated with nuclear reprogramming agents.;The first set of experiments (article 1) focused on determining the expression profile of two growth regulatory imprinted genes, namely Igf2 and H19 in ES cells grown under growth inhibitory conditions, specifically serurn starvation and confluency in addition to in vitro aging (at several passages). Our data showed that culture of ES cells with serum-depleted media and at high cell density increased the expression of both imprinted genes and led to an aberrant methylation profile of a key lgf2 regulatory region (DMR2).;The goal of the second set of experiments (article 2) was to characterize the expression pattern of a group of imprinted genes as well as the methylation profile of DMR2 of lgf2 and relate it to the differentiation status of mouse ES cells exposed to TSA, an inhibitor of histone deacetylases and 5AzaC, a global inhibitor of DNA methylation. In this experiment we report that with the exception of H19, other imprinted genes (Igf2, P57KIP2, Peg1 and lgf2r) expression were upregulated following TSA treatment in ES cells.;The objective of the third experiments (article 3) was to establish the relative expression patterns of several imprinted and non imprinted genes during preimplantation development of fertilized and parthenogenetic mouse embryos cultured in vitro in the presence of TSA and 5AzaC. By the use of quantitative real time PCR analysis we showed that the expression pattern of the imprinted genes Igf2, Peg1, P57KIP2 and Igf2r was upregulated following TSA and 5AzaC treatment.;Finally, the last set of experiments (article 4) was designed to determine whether the cloning procedure is capable of supporting faithful expression of imprinted genes and also whether cloning is able to reprogram imprinted gene expression of donor nuclei (ES and somatic cells) exposed to drugs that modify the levels of histone acetylation and DNA methylation. In this experiment we report that, while some imprinted genes were partially reprogrammed, others remain not fully corrected during development to the blastocyst stage of cloned embryos. (Abstract shortened by UMI.)...