供核细胞对绵羊体细胞克隆效率的影响

【目的】在制作克隆胚胎时,供核细胞直接制约着重构胚的融合数量和发育潜力。研究供核细胞对绵羊体细胞对克隆效率的影响。【方法】从供核细胞的静置时间,研究传代次数与周期处理,供核细胞不同克隆株,不同羊源细胞等,以重构胚融合率和囊胚发育率为检测指标,判定单因素供核细胞对克隆胚胎的影响。【结果】在供核细胞静置时间方面,供核细胞静置15~30 min内完成重构胚制作的融合率极显著高于静置2 h左右的供核细胞组(90.10% vs 78.84%)(P< 0. 01),但囊胚率差异不显著(15.58%vs 14.65%)(P＞0.05);在供核细胞传代次数(d1~d3)和汇合期生长时间方面(24 and 48 h ),各组的融合率差异不显著(70.37%、76.03%、71.92% vs 72.97%)(P＞0.05),但囊胚率有增高的趋势(5.96%、9.06%、15.85% vs 20.16%);在同一供核细胞不同克隆株方面,供核细胞对融合率和囊胚率存在显著(P<0.05)或极显著(P<0.01)的影响;在不同个体方面,供核细胞株对融合率和囊胚率存在极显著的影响(P<0.01)。【结论】15~30 min内的静置供核细胞、传代3次并延长汇合期细胞生长时间(48 h),多选细胞株更有利于克隆胚的制作。

Effect of Donor Cell on the Efficiency of Ovine Somatic Cell Cloning

【Objective】 In the production of cloned embryos, nuclear donor cells directly restrict the fusion number and developmental potential of focused embryos. Therefore, the purpose of this study is to investigate the static time, passage times and cycle treatment of donor cells, different cloned lines of donor cells and different sheep cells, and to determine the effect of single factor donor cells on cloned embryos by using reconstructed embryo fusion rate and blastocyst development rate as detection indexes.【Result】 The fusion rates of pelleted cells on 15-30 minutes in operation liquid drop was higher than longtime (about 2 h) pelleted cells after being removed from plates and re-suspension in centrifugal tube (90.10% vs78.84% )(P<0.01). Embryonic development competence had no significant difference (15.58% vs 14.65%)(P>0.05).Thawed fibroblast cells were cultured one to three passages and to full confluency on the plateau phase of cell growth curve 24 h and 48h, fusion rate of cytoplast-donor cell couplets had no significant difference (70.37%、76.03%、71.92% vs 72.97%)(P>0.05),but embryonic development competence rose gradually(5.96%, 9.06%, 15.85% vs 20.16%); Fusion rates and blastocyst rates of cloned embryos had significant difference(P＜0.05) or remarkable difference (P<0.01). On the different lines from mono adult fibroblast cell; coincidentally, it was same to the different lines from multiple adult fibroblast cell.【Conclusion】 These data showed that static time of resuspension somatic cells among 15-30 minutes in liquid drop, passage several times and prolong the G1 phase of somatic cell formed a confluent monolayer, and multiple somatic cells were conducive to the production of cloned embryos in ovine.