24 IMPROVEMENT OF PORCINE CLONED EMBRYONIC STEM-LIKE CELLS DERIVATION BY ZONA-MEDIATED EMBRYO AGGREGATION

Abstract

Porcine embryonic stem cells (ESC) have become an important model for therapeutic cloning using embryonic stem cells derived by somatic cell nuclear transfer (SCNT). However, embryo quality and blastocyst formation have been major limitations for derivation of cloned embryonic stem-like cells. In this study, we tried to overcome these problems by treating with histone deacetylase inhibitors (HDACi) and aggregating porcine embryos. A porcine embryonic fibroblast (PEF) cell line was used as the source of donor cells injected into the enucleated oocytes. First, to confirm the effect of HDACi in cloned embryo quality, cloned embryos were treated with Scriptaid (histone deacetylase inhibitor). The Scriptaid-treated blastocysts (n = 26) showed significantly increased total cell number (29.50 ± 2.10; P < 0.05) than nontreated blastocysts (n = 21; 22.29 ± 1.50). Then, the cloned embryo quality and blastocyst formation were analyzed in aggregates. Three zona-free reconstructed 4-cell stage SCNT embryos were injected into empty zonae from hatched parthenogenic blastocysts. The blastocyst formation and total cell number of cloned blastocysts was significantly elevated for all the aggregates (76.3% and 83.18 ± 8.33 cells/blastocyst) compared with nonaggregated (31.0%, and 27.11 ± 1.67 cells/blastocyst; P < 0.05). Finally, aggregated blastocysts were cultured on a feeder layer to examine the efficiency of porcine embryonic stem-like cells derivation. Aggregated blastocyst showed higher primary colony formation percentage than nonaggregated cloned blastocysts (20.0 ± 12.3% v. 2.2 ± 1.35%, respectively; P < 0.05). In conclusion, the aggregation of pig SCNT embryos at the 4-cell stage could be a useful technique for improving the development rate and quality of cloned pig blastocyst and derivation efficiency of cloned embryonic stem-like cells.