Drugs that modify the epigenetic?What do they cause to the porcine clones?

MORO L.N; GAMBINI A; SALAMONE D.F; BUEMO CARLA PAOLA; CANEL N

BANGKOK

Congreso; 43nd Annual Conference of the IETS; 2018

Sociedad Internacional de Transferencia de Embriones (IETS).

In this study, we used treatments that modify epigenetic patterns in cloned aggregated embryos. The aim was to improve nuclear reprogramming and to understand the relationship between epigenetic status and the efficiency of SCNT in pigs. Changes in gene expression, in histones and in DNA methylation were studied. An inhibitor of DNA (cytosine 5) methyltransferase (5-Aza) and PD0325901 (PD) as an inhibitor of MEK in the MAPK pathways were used. Maturation of cumulus-oocyte complexes was performed in TCM for 44h at 39°C and 5% CO2. After denudation by treatment with hyaluronidase, mature oocytes were stripped of the zona pellucida using a protease and then they were enucleated by micromanipulation; staining was performed with Hoëchst 33342 to observe metaphase II. Ooplasms were placed in phytohemagglutinin to permit different membranes to adhere between each other; the ooplasm membrane was adhered to a porcine fibroblast from an in vitro culture. Adhered membranes of the donor cell nucleus and enucleated oocyte cytoplasm were electrofused through the use of an electric pulse (80V for 30μs). All reconstituted embryos were electrically activated using an electroporator in activation medium. Then, embryos were incubated in 2mM 6-DMAP for 3h. In vitro culture of zona free embryos was achieved in a well of wells system. Two experimental groups were used, one control group with 3 reconstructed embryo per microwell (3x) and the other group 3x plus the addition of PD + 5 Aza (3x + drugs). Embryos were cultivated for 3 days, then, drugs were washed and continued in culture until day 7 in SOF medium. The drugs (5Aza 1μM + PD 1 μM) modifying the embryonic epigenetics significantly increased the in vitro development rate until the blastocyst stage by embryo, presenting 40.90% of blastocysts in the treated group compared to the control group with a 29 , 41%. It also produced an increase in embryo size, showing statistically significant differences. Regarding to gene expression, their relative expression increased those genes related to cell differentiation as Igf2 and Cdx2; also antiapoptotic genes like Bcl-xl and DNA methylation modulator genes like Mapk1. Pluripotency genes like Oct4 and Nanog were maintained without statistical differences. The Bax proapoptotic gene significantly decreased its expression with drug treatment as did the Klf4 gene (T student´s test p