C57BL/6J小鼠冻融IVF原核胚用于Cas9显微注射探讨  

Frozen-thawed pronuclear embryos by in vitro fertilization of C57BL/6J mice can be used for Cas9 microinjection

作　　者：曾文滔 周建丽[1] 赵书琴[1] 张亚男[1] 梅云[1] 

机构地区：[1]南京医科大学医药实验动物中心,南京211166

出　　处：《中国比较医学杂志》2017年第8期66-69,共4页Chinese Journal of Comparative Medicine

摘　　要：目的探讨小鼠体外受精(IVF)冻融后用于Cas9注射的可行性。方法新鲜C57BL/6J小鼠卵子IVF后采用冻存管法(EFS20/40)冷冻原核胚(单细胞胚)和2细胞胚胎,次日复苏后培养。比较冻融原核胚和2细胞胚胎的回收率及成活率。选取部分冻融原核胚与新鲜原核胚同时进行Cas9和稀释液胞质注射并培养至2细胞,分别比较注射后的存活率及2细胞发育率。结果冻融后的B6小鼠原核胚的回收率为92.5%、成活率为92.8%,2细胞胚的回收率为90.5%、成活率95.8%,两组数据差异无显著性(P>0.05)。新鲜原核胚Cas9注射后的存活率为92.7%,空白组注射组为97.5%,冻融原核胚Cas9注射后的存活率为82.6%,空白组为92%,冻融组Cas9注射与各组差异有显著性(P<0.05),其它各组差异无显著性(P>0.05)。注射后的2细胞胚发育率差异无显著性(P>0.05)。结论冻融原核胚可用于Cas9显微注射。Objective To verify the feasibility of Cas9 microinjection in frozen-thawed pronuclear embryos,based on the model of pronuclear embryos of C57BL/6J mice by in vitro fertilization. Methods After fertilized mouse pronuclear embryos cultured in vitro,one-cell and 2-cell embryos were frozen using EFS20/40 cryopreservation tube. The next day recovered and then cultured. The recovery rate and survival rate of the one-cell and 2-cell embryos were compared. The frozen-thawed and fresh pronuclear embryos were injected with Cas9 mixture and injection buffer into the cytoplasm,and then cultivated to 2-cell embryos,and the survival rate and development rate of the 2-cell embryos were compared. Results The recovery rate of frozen-thawed one-cell embryos was 92. 5%,the survival rate was 92. 8%,the recovery rate of 2-cell embryos was 90. 5% and the survival rate was 95. 8%,showing no significant difference. Furthermore,the survival rate of fresh one-cell embryos after Cas9 injection was 92. 7%,the survival rate of one-cell embryos of the blank group was97. 5%. While the survival rate of Cas9 injected frozen-thawed one-cell embryos was 82. 6%,and that of the blank group was 92%,showing a significant difference between the frozen-thawed injected group and other groups（ P〈0. 05）. The development rate of 2-cell embryos after Cas9 injection was not significantly different. Conclusions Frozen-thawed pronuclear embryos can be used for Cas9 microinjection.

关 键 词：体外受精 受精卵 胚胎冷冻 Cas9 显微注射 

分 类 号：R-33[医药卫生]