绵羊体细胞核移植融合效率影响因素的研究

The Effect of Receptor,Donor and Pulsed Strength on The Fusion Rate of Somatic Cell Nuclear Transfer in Ovine

【目的】提高克隆胚胎融合率。【方法】从受体卵母细胞的细胞松弛素处理组与对照组,供体细胞的梯度差速选择(胰酶浓度0.1%、0.25%,消化时间3 min、1 min)和4组电脉冲融合参数(100 V、120 V、130 V、150 V)等3个环节对绵羊体细胞核移显微操作进行了优化,以重构胚胎的融合率和卵裂率作为检测指标。【结果】未经细胞松弛素的卵母细胞组融合率显著高于试验组(P<0.05),而胚胎卵裂率差异不显著(P>0.05);胰酶梯度差速消化供体细胞具有初选作用,各组融合率为71.71%、75.86%、78.75%、75.56%,融合压为130 V时融合率最高。【结论】优化后的程序更适合克隆胚胎的膜融合。

【Objective】Most current studies of nuclear transfer in mammalian oocytes have used electrofusion to incorporate donor cell nuclei into enucleated oocyte cytoplast. However,the fusion was hampered by bad incorporate of donor and receptor which play an important role in somatic cell nuclear transfer. The purpose of this project aims to improve the fusion of somatic cell nuclear transfer in ovine. 【Method】Three experiments to optimize the nuclear transfer proceduces were tested: the oocytes used as receptor cells were treated with cytochalasins B in the experiment group( CB group) but not in the control group( NC Group),and the trypsinized fibroblasts were used as donor cells in different times and concentrations,four parameters of pulsed strength( 100V,120V,130V,150V) were selected to fuse the SCNT couplets. Fusion rate and cleavage rate were evaluated during the SCNT procedure.【Result】These results suggested that the fusion rate of NC group was higher than CB group( 78. 35% vs 62. 09%),but cleavage rates had no significant difference( 76. 39% vs80. 26%). The different concentrations( 0. 1%,0. 25%) and times( 3min,1min) of trypsin would primarily screen out excellent donor cells. The fusion rate of parameter 130V was the highest among of the four groups( 71. 71%,75. 86%,78. 75% and 75. 56%). 【Conclusion】The optimized procedure was more efficient to fuse the SCNT membrane of couplets.