不同电融合和激活参数对猪体细胞核移植胚胎发育的影响

Influence of different parameters of electrical fusion and activation on development of porcine somatic cell nuclear transfer embryo

目的:摸索猪体细胞核移植最佳的融合和激活条件。方法:利用体外成熟的去核猪卵母细胞为受体,颗粒细胞为供核细胞,透明带间隙注射法构建重构胚,对电融合和激活的参数(电场强度、脉冲宽度、脉冲次数、融合液中Ca2+浓度、联合激活参数)进行摸索。结果:发现在电场强度1.5 kV/cm,脉冲宽度30μs,2次脉冲(间隔3 s),电融合液中0.1 mmol/L的Ca2+浓度下进行电激活,进而用(CB,7.5μg/m l)+(6-DMAP,2 mmol/L)激活3.5 h发育率最高,48 h卵裂率为72.5%,第6天桑葚胚率为35.1%;初次尝试了在胚胎培养前48 h加入0.05 mol/L的甘露醇来增加渗透压以减少电激活后重构胚的碎片化程度,但发现卵裂率及桑葚胚发育率与对照组差异不显著。结论:初步得到了适宜的猪体细胞核移植的融合和激活条件,为随后克隆胚早期发育基因的研究打下了基础。

Objective： To explore the best conditions of electrical fusion and activation influencing the development of porcine somatic cell nuclear transfer embryo. Methods： The oocytes matured in vitro for 40 - 42 h were enucleated as the recipient cytoplasts and the cumulus cells served as the nuclear donor cells, which were inserted into the perivitelline space of the enucleated oocyte to form the reconstructed couplet. Five important factors which were electrical field strength, pulse number, pulse width,Ca^2＋ concentration in the fusion medium and electrical activation combined chemical activation were studied. Results： The best parameter was as follows ： electrical field strength 1.5 kV/cm, pulse width 30μs, pulse number 2 times （3s interval）, 0.1 mmol/L Ca^2＋ concentration in the fusion medium. After the electrical activation, the couplets were further activated in CB（7.5μg/ml） ＋6-DMAP（2 mmol/L）for 3.5 h. The cleavage rate at 48 h was 72.5% ,and the morula rate on the 6th day was 35.1%. It was the first time to try supplementing the mNCSU-23 ＋ 10% FBS with 0.05 mol/ L mannitol for the first 48 h increasing osmolarity to suppress fragmentation, but it was found that the cleavage rate and developmental rate to the morula stage were not significantly different from the control group. Conclusion：The optimal condi- tions for fusion and activation of the porcine somatic cell nuclear transfer were obtained preliminarily, making the research on early development genes of the cloned porcine embryo possible.