水牛受精卵胞质内注射转基因的初步研究

Study on Transgenesis by Cytoplasmic Injection of Exogenous DNA into Buffalo Zygote

[目的]旨在探讨通过向水牛受精卵胞质内注射外源DNA实现转基因的可行性。[方法]水牛卵母细胞体外成熟20～22 h后随机分为2组:①在体外受精7～10 h或18～20 h后向卵胞质内注入约7.5 pl 50μg/ml含线性EGFP片段的DNA溶液;②分别注入单个精子与约7.5 pl 50μg/ml含线性EGFP片段的DNA混合物,观察外源基因在胚胎发育过程中的表达情况。[结果]受精卵胞质内注射的早期胚胎基因表达率、囊胚基因表达率与ICSI-Tr差异不显著(P>0.05),且IVF 7～10 h时注射的分裂率、早期胚胎基因表达率均显著高于18～20 h(P<0.05)。[结论]水牛IVF受精卵胞质内注射外源基因能获得转基因胚胎,且IVF后7～10 h注射的效果优于IVF后18～20 h注射。

[Objective] This study aimed to investigate the feasibility of transgenesis by injecting exogenous DNA into zygote cytoplasm of buffalo.[Method] Buffalo oocytes were randomly divided into two groups 20-22 h after in vitro maturation.One group of oocytes was introduced with about 7.5 pl of 50 μg/ml DNA solution containing linear EGFP fragment by cytoplasmic injection 7-10 h or 18-20 h after in vitro fertilization（IVF）;the other group of ooctyes was introduced with mixture of a single buffalo sperm and about 7.5 pl of 50 μg/ml DNA solution containing linear EGFP fragment by cytoplasmic injection（generally called ICSI-Mediated Gene Transfer,ICSI-Tr）.Expression of exogenous DNA was observed and recorded during the process of embryonic development.[Result] Early embryonic gene expression efficiency and blastocyst gene expression efficiency in IVF group showed no significant difference compared with that in ICSI-Tr group（P0.05）.In addition,the cleavage rate and early embryonic gene expression efficiency in IVF group were significantly higher with injection at 7-10 h post IVF than 18-20 h post IVF（P0.05）.[Conclusion] These results indicate that transgenic buffalo embryos can be generated by injecting exogenous DNA into cytoplasm of IVF oocytes,and the optimal injection time is 7-10 h post IVF.