Pigs provide outstanding models of human genetic diseases due to the striking similarities to human anatomy, physiology and genetics. Pig induced pluripotent stem cells (piPSCs) have been generated for several years but the cloning efficiency using unsynchronized piPSCs was extremely low. Here, we reported a method to produce diploid cloned embryos from piPSCs which were synchronized to metaphase. The piPSCs cell line was established using a drug-inducible system and exhibit similar morphology to mouse embryonic stem cells with normal karyotype. After synchronized by a two-step block method with aphidicolinand nocodazole, 77.6% of the cells were arrested at G2/M phase. Round cells ranged from 1719 μm were selected and fused with enucleated MII oocytes. After activation, 81.3% of reconstructed embryos extrudedone pseudo-second polar bodies (p2PB). The immunofluorescent results confirmed that half chromatids were extruded with the p2PB. However, 2 mM 6DMAP treatment post activation blocked the p2PB extrusion. Moreover, immediately activation method yielded significantly more blastocysts than delayed activation (31.3 vs 16.0%, based on fused embryos). 6DMAP treatment post p2PB extrusion also didnt improve the blastocyst formation rate. Karyotyping of the blastocysts indicated that 61.1% blastocysts were diploid. This study demonstrated a new efficient way to produce cloned embryos from piPSCs which synchronized to mitotic metaphase. Further studies will be focused on the pluripotent gene expression of these embryos and the feasibility of using these embryos to produce cloned pigs.
This work was supported by a grant from the National Research Foundation of Korea Grant Government (NRF-2013R1A1A2064750), Republic of Korea.
02 - 04 Sep 2014
World Congress of Reproductive Biology