【摘要】 目的　原核胚是转基因等生物技术所必需的主要材料 ,其冷冻保存使操作不受时间和空间的限制。另外 ,冷冻保存还可以避免体外培养过程中的细胞发育阻断期。方法　在室温 (2 0℃或 2 5℃ )条件下 ,以乙二醇、DMSO为主体抗冻保护剂配制成的玻璃化溶液 (EFS、EDT、EDFS) ,不借助冷冻仪 ,对小鼠原核胚进行一步法和二步法玻璃化冷冻保存。结果　 2 0℃室温条件下 ,用EDFS4 0平衡 1min一步法冷冻解冻后的原核胚 ,经培养后囊胚发育率最高仅为 4 7% ,与新鲜原核体外培养的对照组 (75 % )的差异极显著 (P <0 0 1) ;当原核在 10 %E +10 %D溶液中预处理 5min ,移入EDFS30中平衡 30s二步法冷冻保存 ,解冻后的囊胚发育率达 6 8%。而室温升至 2 5℃ ,二步法冷冻保存后原核胚的囊胚发育率高达 77% ,与对照组差异无显著性 (P >0 0 5 )。用最佳冷冻组的原核胚或解冻后培养到囊胚移植给受体母鼠均获得产仔。结论　本研究对小鼠原核胚实施玻璃化冷冻保存 ,经体外培养和移植结果与对照组无显著性差异 ,证明了本方法的可行性更多还原

【Abstract】 Objective Mouse zygotes are the main materials for mouse transgenic technology by microinjection. The successful zygotes cryopreservation could make this technology beyond spacial and time limitations, and avoid the problem of in vitro developmental block. Methods In this experiment, mouse zygotes were cryopreserved by vitrification with one step or two step methods using vitrification solutions (EFS,EDT,EDFS) at room temperature (20 or 25℃). Results The zygotes were equilibrated in EDFS40 for 1 min and plunged into nitrogen liquid (one step method) at 20℃. The blastocyst rate was 47% in vitro culture after thawing. The rate was significantly lower than that of the control (75%)( P <0 01). However, when zygotes were pretreated in the 10%E+10%D for 5 min and then equilibrated for 30s in EDFS30 (two step method), followed by plunging into liquid nitrogen, the blastocyst rate increased to 68% after thawing and in vitro culture. When the zygotes were cryopreserved by the two step method at 25℃, the blastocyst rate reached 77%, it was not significantly different from that of the control ( P >0 05). Live pups were born after embryo transfer either from vitrified thawed zygotes or blastocysts from vitrified thawed zygotes. Conclusion These results demonstrated that our vitrification methods were available for cryopreservation of mouse zygotes.更多还原