86 CREATION OF PORCINE TRANSGENIC NUCLEAR-TRANSFERRED EMBRYOS RECONSTRUCTED WITH EGFP-EXPRESSING ADULT DERMAL FIBROBLAST CELLS ANALYZED ON APOPTOSIS

Abstract

The purpose of our study was to determine the in vitro developmental competences of porcine nuclear transfer (NT) embryos reconstructed with pWAPhGH-GFPBsd transgene-nucleofected gilt ear skin-descended fibroblast cells, which had been diagnosed on apoptosis through the live-plasma membrane fluorescent tagging. Frozen–thawed fibroblast cells, which had been in vitro-cultured up to a total confluency after 2–8 passages, were used for analysis. To detect the early apoptotic changes in the fibroblast cells, single nuclear donor cell suspension was labeled with the conjugate of Annexin V and eGFP protein. The source of recipient cells were in vitro-matured oocytes. Maternal chromosomes were eliminated by a chemically assisted microsurgical technique. Fibroblast cell–ooplast couplets were simultaneously fused and activated. Reconstructed embryos were cultured in NCSU-23/BSA/FBS medium for 6–7 days. The rates of cleavage and development to morula/blastocyst stages were examined on Days 2 and 6/7, respectively. After fluorescent analysis of adult dermal fibroblast cells, it was shown that a relatively high proportion (ranging from 20 to 30%) of donor cells exhibited ultrastructural late-apoptotic or necrotic changes. In contrast, from among the morphologically normal cells, an extremely low rate (ranging from 0 to 2%) of the cells emitted the Annexin V-eGFP-derived green fluorescence, but the other ones did not emit this biochemiluminescence. This suggests that the former subpopulation of the cells was early-apoptotic, and the latter was non-apoptotic. A total of 158 enucleated oocytes were successfully fused with non-apoptotic transgenic nuclear donor cells and intended to be in vitro-cultured. Out of 158 reconstructed oocytes, 106 (67.1%) NT embryos were cleaved. The frequencies of cloned embryos that reached the morula and blastocyst stages were 48/158 (30.4%) and 21/158 (13.3%), respectively. In conclusion, the nucleofection efficiency of in vitro-cultured porcine dermal fibroblast cells as estimated by nuclear donor live-fluorescent evaluation based on the expression index of the eGFP reporter transgene was nearly 100%. Moreover, our results demonstrate that the morphological criteria commonly used for cell viability classification are a sufficient selection factor for qualitative evaluation of nuclear donor cells to somatic cell cloning. It was also found that porcine nuclear-transferred morulae and blastocysts exhibited an approximately 100% index of xenogeneic eGFP gene transcriptional activity, which revealed the live diagnostics of emission intensity for green fluorescent protein-derived biochemiluminescence.

This research was supported by the State Committee for Scientific Research as a Solicited Project number PBZ-MIN-005/P04/2002/6 from year 2003 to year 2006.