关键词: 细胞周期, 体细胞核移植, 猪

Abstract: This study was undertaken to systematically examine the effects of different donor cells and numbers of pas-sages on the development of nuclear-transferred porcine embryos, so as to establish a preliminary procedure for porcine cloning. Porcine oocytes obtained at slaughter were matured in vitro for 40―44 h and then enucleated in manipulation me-dium containing 5 μg/mL cytochalasin B. Fibroblast cells (FC), oviduct epithelial cells (OEC), granulosa cells (GC) and cumulus cells (CC) after 3―9 passages in 10% FBS-supplemented culture medium were either treated by serum starvation (0.5% FBS for 2―9 days), 0.1 μg/mL aphidiconlin (APD) for 1 day and 0.5% FBS for 2―9 days or left untreated in com-plete medium for 2―9 days. They were transferred into enucleated oocytes by microinjection or electric fusion (100 V/mm, 30 μs and 1 pulse). Reconstituted embryos were activated with a combination of calcium ionophore A23187 or electric pulse and 6-DMAP, and cultured for 6 days, to evaluate their cleavage and embryonic development. The cleavage rate of embryos reconstructed with FC and GC pretreated with 0.1 μg/mL APD + 0.5% FBS were significantly higher than that of serum starvation group and control group (P<0.01). There was a significant difference in the cleavage rate and embryonic development among embryos derived from GC, CC and FC, OEC pretreated with 0.1 μg/mL APD + 0.5% FBS. The cleav-age rate of embryos reconstructed with GC by electrofusion was significantly higher than that by microinjection (P<0.05), but no difference was found in the proportion of embryos that developed to blastocysts. About 75% to 85% of GC at 3 and 6 passages, and FC at 6 and 10 passages had a normal karyotype, and resulted in similar cleavage rate and blastocyst devel-opment. These results indicate that: (1) FC and GC can be cultured up to 9 passages and maintain a relatively stable karyo-type; (2) Treatment of donor cells with 0.1 μg/mL APD prior to nuclear transfer can improve the efficiency of somatic cell nuclear transfer in buffalo but serum starvation is inefficient in our system; (3) Both FC and GC cells can be used as the donor karyoplasts for nuclear transfer, and their efficiency is not influenced by the culture passages. (4) The development of reconstructed embryos by electrofusion is higher than that by microinjection, but there is no difference in the overall effi-ciency between the two methods.