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The ibex ( Capra ibex ) is currently listed as a priority protected animal by the Chinese government. Due to the limited availability of ibex oocytes, the objective of this study was to explore the feasibility of using domestic goat ( Capra hircus ) cytoplasts to reprogram nuclei from ibex donor fibroblasts in interspecies nuclear transfer (iSCNT). An effort was thus made to produce ibex embryos via iSCNT. The trial was performed using three treatment groups. The embryo cleavage rates on day 3 of in vitro culture and the blastocyst development rates on day 7 were recorded. In Treatment A, the female ibex fibroblasts were used as donor cells and of the fused cell-cytoplast couplets, 68% cleaved and 11% reached the blastocyst stage. In Treatment B, domestic adult dairy doe fibroblasts were used as donor cells and as comparison of homologous nuclear transfer (NT). Of fused cell-cytoplast couplets, 76% cleaved and 31% reached the blastocyst stage. In Treatment C, the development of parthenogenetic goat oocytes was used as the control and 90% of the embryos cleaved and 43% reached the blastocyst stage. No significant difference was found between treatment groups A and B in embryo cleavage rate, but a significant difference ( P P C . hircus ) cytoplasts supported the mitotic cleavage of ibex karyoplasts and were capable of reprogramming the nucleus to achieve a blastocyst stage embryo in exotic Capra which has the potential of alleviating species on the endangered list.

In this study, we introduced a co-transfection method for the selection of donor nuclei in somatic cell-mediated nuclear transfer. Two vectors were constructed in our experiment. One was pMSCV-GFP carrying the neomycin-resistant gene (Neo(r)) and the green fluorescent protein (GFP) reporter gene; the other was pBC1-GFP carrying the mammary gland-specific promoter and target gene GFP. Ovine adult fibroblasts were co-transfected with pMSCV-GFP and pBC1-GFP. The data from this work demonstrated that the GFP genes in both vectors could successfully co-integrate into the genomes of ovine adult fibroblasts in three of the four transgenic cell clones assayed. Furthermore, PCR analysis of transgenic embryos proved that the GFP genes in both vectors could co-integrate into the genomes of the reconstructed embryos. Subsequently, analysis of the developmental rate of the reconstructed embryos after nuclear transfer indicated that the blastocyst rate from the co-transfected donor cells was similar (approximate 8 percent) to that from individual pMSCV-GFP transfected donor cells. The influence of co-transfection resulting in modification of donor nuclei on development of reconstructed embryos was also investigated. The results of flow cytometric analysis indicated that the co-transfected ovine fibroblasts had similar quiescent characteristics in terms of cell cycle (G0+G1 percent: 73.20 +/- 4.04) to the individual pMSCV-GFP transfected fibroblasts (G0+G1 percent: 70.77 +/- 1.19) after they were treated with serum starvation for five days. Our results suggest that the co-transfection method can be used for selection of donor cell clones in somatic cell-mediated gene transfer experiments. It can be potentially extended to applications related to expression of functional protein in mammary glands and other transgenic research relevant to nuclear transfer.