小鼠第一极体基因组移植的时间窗探索

Study on the time-window of mouse first polar body genome transfer

目的以杂交一代(BDF1)小鼠为模型,探索小鼠第一极体基因组移植(PB1T)的最佳时间窗,提高第一极体基因组移植重构胚存活率和体外发育能力,为临床研究和应用提供前期动物实验基础。方法获取注射人绒毛膜促性腺激素(h CG)后12.5、13.5、14.5、15.5 h 4个时间点的MII卵子作为实验组。分别取第一极体作为核供体,取h CG后14h的MII卵子,去除纺锤体染色体复合物后作为胞质供体,构建四组第一极体基因组移植重构胚。获取MII卵子进行体外受精并培养作为对照组(IVF组)。体外培养并观察实验组4组重构胚以及对照组卵子存活情况、体外受精、卵裂和囊胚发育情况。结果在4组实验组中,13.5 h组的PB1T重构卵子的存活率、体外受精率、2-细胞形成率、4-细胞形成率和囊胚率均最高,与IVF对照组相比差异无统计学意义(P>0.05)。14.5 h组的重构卵子的存活率、体外受精率和后续发育率稍低于13.5 h组,与对照组相比差异无统计学意义(P>0.05)。15.5 h组的重构胚体外受精率和囊胚形成率均最低(76%、41.7%),与对照组相比差异有统计学意义(P=0.020 0,P=0.005 7)。结论 BDF1小鼠h CG后13.5 h和14.5 h的卵子的第一极体基因组在新的供体胞质中能够更好地重建发育潜能,行PB1T可能的最佳时间窗为h CG后13.5~14.5 h。

Objective To explore the optimal time window of the first polar body genome transfer（ PB1 T） in B6 D2 F1 mouse（ BDF1）,to improve the survival rate and in vitro development ability of the PB1 T-embryos,and to provide the basis of preclinical animal experiment for clinical research and application. Methods Experiment group： About 12. 5,13. 5,14. 5,15. 5 hours after the h CG injection,the MII oocytes from oviducts were obtained,respectively. The first polar body was collected as nuclear donor and enucleated MII oocytes,about 14 hours after the h CG injection,were collected as cytoplasm donor. PB1 T-embryos were reconstructed and cultured in vitro. Control group（ IVF group） ： The MII oocytes were obtained and cultured in vitro. Survival rate after micromanipulation,fertilization rate,cleavage rate and blastocyst formation rate were analyzed for the five groups.Results The survival rate,fertilization rate,2-cell rate,4-cell rate and blastocyst rate of the PB1 T-embryos in13. 5-hour group were the highest among the four experiment groups,but there was no significant difference（ P〉 0. 05） compared with the IVF group. In the 14. 5-hour group,the survival rate,fertilization rate and subsequent development rates were slightly lower than that in 13. 5-hour group,and there was no significant difference compared with the IVF group（ P〉0. 05）. The fertilization rate and blastocyst rate in 15. 5-hour group（ 76. 0%,41. 7%） were significantly lower than that in control group（ P = 0. 020 0,P = 0. 005 7）. Conclusion In BDF1 mouse,the first polar body genome of 13. 5 hours and 14. 5 hours after the h CG injection can better support the development of reconstructed embryos. The period of 13. 5 ~ 14. 5 hours after the h CG injection may be the best time window for PB1 T.