猪克隆胚胎激活方法优化与转人溶菌酶基因克隆猪的生产

Optimizing of Activation Protocols and Production of Transgenic Pigs Expressing Human Lysozyme by Somatic Cell Nuclear Transfer

为了提高转基因克隆效率和获得转人溶菌酶基因克隆猪,研究了不同电激活参数和化学辅助激活方法对猪克隆胚胎和孤雌胚胎体外发育的影响.结果发现:电场强度会显著影响克隆胚胎的融合率和体外发育能力(P<0.05),电脉冲次数对克隆胚胎体外发育促进作用不显著(P>0.05),而相同电激活条件下克隆胚胎和孤雌胚胎的体外发育能力变化趋势不同;电激活后再利用放线菌酮+细胞松弛素B(CHX+CB)处理4 h能显著提高克隆胚胎的囊胚率(P<0.05),而用二甲基氨基嘌呤(6-DMAP)处理没有提高克隆胚胎囊胚率(P>0.05),但6-DMAP或CHX+CB处理均可显著提高孤雌胚胎的囊胚率(P<0.05).上述结果表明,最佳的孤雌激活条件并不一定是克隆胚胎的最佳激活条件.本研究中猪克隆胚胎的最佳激活方法为1.6 kV/cm、100μs、2次直流电脉冲间隔100μs,再辅以CHX+CB处理4 h.利用优化的激活条件成功获得了乳腺特异表达人溶菌酶的转基因猪,为猪转基因育种奠定了基础.

In order to improve the cloning efficiency and obtain human lysozyme（hLY） gene transgenic pigs,the present study was carried out to investigate the effects of different electric activation parameters and chemicals on in vitro development of embryos derived from parthenogenesis（PA） and somatic cell nuclear transfer（SCNT）.The results showed that the electric strength could influence the fusion rate and developmental ability of SCNT embryos（P 0.05）,and number of electric pulses had no significant effect on SCNT embryos development（P 0.05）,yet different variation tendency was found in developmental ability between PA and SCNT embryos under same activation parameters.The blastocyst rate of SCNT embryos was improved when treated with CHX＋CB for 4 h after electric activation（P 0.05）,whereas 6-DMAP did not（P 0.05）.On the contrary,either CHX＋CB or 6-DMAP treatment after electric pulses could improve the blastocyst rate of PA embryos,indicating that the best activation method for PA was not necessarily the best for SCNT.The best activation protocol of SCNT embryos in our study is two pulses of 100 μs,direct current of 1.6 kV/cm electric strength with 100 μs interval,and followed by CHX＋CB treatment for 4 h.With the activation protocol,the mammary gland expressed hLY transgenic pigs were generated.It could help for improving piglets survival rate in transgenic breeding.