
%0 Journal Article
%A Skrzyszowska, M. 
%A Samiec, M. 
%A Smorag, Z. 
%A Lipinski, D. 
%A Slomski, R. 
%T 86 CREATION OF PORCINE TRANSGENIC NUCLEAR-TRANSFERRED EMBRYOS RECONSTRUCTED WITH EGFP-EXPRESSING ADULT DERMAL FIBROBLAST CELLS ANALYZED ON APOPTOSIS
%J Reproduction, Fertility and Development
%D 2006/12/12
%V 19
%N 1
%P 160-161
%K 
%R https://doi.org/10.1071/RDV19N1AB86
%U https://www.publish.csiro.au/paper/RDV19N1AB86 
%X 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&#8211;thawed fibroblast cells, which had been in vitro-cultured up to a total confluency after 2&#8211;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&#8211;ooplast couplets were simultaneously fused and activated. Reconstructed embryos were cultured in NCSU-23/BSA/FBS medium for 6&#8211;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. 
