PARENT SESSION
GENE REGULATION AND FUNCTION IN THE REPRODUCTIVE AXIS
Kent
7:30 AM-10:00 AM

(361) ABEARANT EXPRESSOIN PROFILE OF IMPRINTED GENES IN CLONED MOUSE EMBRYOS RECONSTRUCTED WITH ES CELLS TREATED WITH 5AzaC OR TSA.

Baqir, Senan¹, Qi, Zhou², Renard, Jean-Paul², Smith, Lawrence¹, ¹ Centre de Recherche en Reprodution Animale (CRRA), St-Hyacinthe, CA² Unité de Biologie du Developpement et Biotechnologies, Jouy-en-Josas, FR

ABSTRACT- Growth abnormalities associated with mice derived by nuclear transfer may be caused by epigenetic deregulation of imprinted genes due to abnormal resetting of methylation and acethylation patterns during development. Our objective is to determine whether the cloning procedure is able to reprogram expression of imprinted genes of donor nuclei exposed to drugs that alter DNA methylation and histone acetylation. To this end, ES cells were treated with either TSA (histone deacetylase inhibitor) or 5AzaC (DNA demethylation agent) prior to injection into enucleated oocytes. Both drugs have previously been shown to overexpress imprinted genes in ES cells. RNA samples were extracted from morula and blastocyst-stage embryos, reversed transcribed and the expression pattern of imprinted genes (Igf2 and P57^KIP2) and a housekeeping gene (Gapdh) was examined by PCR. Samples were standardized with an exogenous control (Globin) and expressed as fold changes in relation to non-cloned in vitro produced embryos. Although Gapdh expression did not differ among treated and non-treated groups, the expression level of Igf2 and P57^KIP2 was higher in cloned embryos produced from TSA-treated ES donor cells (3.5 and 2.3 fold) compared to embryos cloned from non-treated ES cells (1.4 and 1.2 fold). Similarly, cloned embryos reconstructed with ES cells treated with 5AzaC overexpressed Igf2 and P57^KIP2 (2.1 and 2.5 fold) compared to embryos cloned from non-treated ES cells (1.3 and 1.8 fold). These results indicate that imprinted genes are not correctly reprogrammed during development to blastocyst in cloned embryos. Moreover, cloned blastocyst derived from ES cells treated with TSA and 5AzaC show additional alteration of imprinted gene expression, suggesting that improper resetting of genetic program after nuclear transfer is directly related to altered methylation and acetylation patterns (Funded by NSERC of Canada).

KEY WORDS: Embryo Cloning, imprinting, Acetylation, Methylation