腺病毒介导的高效生产水牛转基因胚胎条件的摸索

doi:10.16431/j.cnki.1671-7236.2019.03.015

摘要/Abstract

摘要：

试验旨在探究腺病毒感染水牛颗粒细胞和胚胎的最佳条件，以提高转基因水牛胚胎的生产效率。以水牛颗粒细胞和体外发育的胚胎为研究对象，分别用0、1×10⁰、1×10^-1、1×10^-2、1×10^-3、1×10^-4、1×10^-5 GFU/mL腺病毒感染水牛颗粒细胞，获得最佳感染浓度后，在最佳浓度条件下分别感染24、48、72、96 h，摸索最佳感染时间，用倒置荧光显微镜观察试验结果。利用腺病毒感染水牛颗粒细胞的最佳浓度和时间分别感染2细胞和4细胞期胚胎，对胚胎的最佳感染条件进行摸索，分析胚胎分裂率、囊胚率和转染囊胚率。结果显示，1×10^-2 GFU/mL浓度和48 h感染时间可获得水牛颗粒细胞的最佳腺病毒感染效率。腺病毒感染2细胞和4细胞期无透明带和非完整透明带胚胎后胚胎发绿光，而感染完整透明带胚胎后不发光，2细胞期胚胎感染后停止发育，4细胞期开始感染的非完整透明带胚胎可继续发育至囊胚。于4细胞期分别感染完整透明带组、非完整透明带组和无透明带组水牛胚胎，结果显示，非完整透明带转染组在囊胚率和囊胚转染效率上均优于其他组。综上，非完整透明带，1×10^-2 GFU/mL和48 h为感染浓度和时间，4细胞期为感染起始期的腺病毒介导的水牛转基因胚胎生产方法能够实现目标基因在水牛胚胎中的高效表达，从而达到提高转基因水牛胚胎生产效率的目的。

关键词: 腺病毒; 水牛; 转基因

Abstract:

In order to improve the production efficiency of transgenic buffalo embryos,this study was aimed to explore the optimal conditions of adenovirus-infected buffalo granular cells and embryos.Taking buffalo granule cells and embryos developed in vitro as research objects,buffalo granular cells were infected with 0,1×10⁰,1×10^-1,1×10^-2,1×10^-3,1×10^-4,1×10^-5 GFU/mL adenovirus to obtain the best infection concentration;The buffalo granular cells were infected with the optimum concentration condition for 24,48,72 and 96 h to obtain the optimal infection time,and the results were observed by inverted fluorescence microscope.The optimal concentration and time of adenovirus-infected to buffalo granular cells were infected to the embryos in 2- and 4-cells stages,respectively,the optimal conditions of embryo infection were explored,and the embryo cleavage rate,blastocyst rate and transfection blastocyst rate were analyzed.The results showed that the optimal adenovirus infection efficiency of buffalo granular cells could be obtained by 1×10^-2 GFU/mL concentration and 48 h infection time of adenovirus.The embryos in 2- and 4-cells stages without zona pellucida and non-complete zona pellucida were infected with adenovirus got green light,while there was no light in the complete zona pellucida embryo.The embryos in 2-cell stage stopped developing after infection,while the incomplete zona pellucida embryos in 4-cell stage could continue developing into blastocyst.The blastocyst rate and blastocyst transfection efficiency of incomplete zona pellucida group was superior to other groups after infected with adenovirus in 4-cell stage.In summary, the incomplete zona pellucida,1×10^-2 GFU/mL,48 h,and 4-cell stage infection contributed to efficient expression of the target gene in buffalo embryo,and then improved the efficiency of transgenic buffalo embryo production.

Key words: adenovirus; buffalo; transgenes

中图分类号:

S814.8

陆杏蓉, 庞春英, 邓廷贤, 段安琴, 马小娅, 梁莎莎, 梁贤威. 腺病毒介导的高效生产水牛转基因胚胎条件的摸索[J]. 《中国畜牧兽医》, 2019, 46(3): 764-773.

LU Xingrong, PANG Chunying, DENG Tingxian, DUAN Anqin, MA Xiaoya, LIANG Shasha, LIANG Xianwei. Condition of Adenovirus-mediated High Efficient Transgenic Embryo Production in Buffalo[J]. , 2019, 46(3): 764-773.

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