O-7: Improved In Vitro Development of Cloned Bovine Embryos Using S-Adenosylhomocysteine,A Non-Toxic Epigenetic

Background: Development of cloned bovine embryos. Materials and Methods: Oocytes collection,oocyte denudation, oocyte enucleation, nuclear transfer, electrofusion, reconstructed embryo activation, culture of reconstructed and IVF embryo,and treatment with SAH post fusion interval Treatment of reconstructed embryos with TSA for 12 hours after activation, preparation of somatic donor cells, donor cells transfectionwith GOF18-DPEEGFP plasmid containing neomycin resistance genes via lipofection method, cell cycle synchronization, EGFP- POU5F1 fluorescence, immunofluorescence staining quantitative real time RT-PCR. Results: The results of this study indicated that post-fusion treatment with SAH has a time dependent effect on DNA-methylation and histone-acetylation, developmental competence and gene expression of the cloned embryos. In addition, these results might improve quality of cloned bovine embryos to produce transgenic cattle. Conclusion: The results of this study indicated that post-fusion treatment with SAH, a non-toxic DNMTs inhibitor, has a time dependent effect on DNA-methylation and histone-acetylation, developmental competence, and gene expression of the cloned embryos. Although this effect has been shown to be time dependent, there was not a consistent effect of a certain SAH interval on all assessed aspects of embryo development. Accordingly, while 12 hours treatment significantly increased blastocyst rate of cloned embryos compared with IVF and control SCNT, 48 hours treatment with SAH produced embryos resembling to IVF embryos in terms of DNA-methylation status, fluorescent intensity for EGFP and POU5F1 gene expression. Even though compromised results of 72 hours treatment with SAH strongly indicated that any treatment with SAH should be terminated before the critical stage of de novo methylation. To make a final conclusion on the impact of SAH treatment time interval, further studies on full term development of the resultant cloned embryos should be performed. These results might improve quality of cloned bovine embryos to produce transgenic cows because there are potentially large profits to be made from transgenic pharmaceutical productions as F-VIII, F-IX, Protein C, AT III, Fibrinogen, Albumin and Activase® (Alteplase) or t-PA factors