水牛受精卵胞质内注射转基因的初步研究(英文)

Preliminary Study on Transgenesis by Injecting Exogenous DNA into Zygote Cytoplasm of Buffalo

[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20～22h后随机分为2组,①在体外受精7～10或18～20h后向卵胞质内注入约7.5pL50μg/ml含线性EGFP片段的DNA溶液;②则分别注入单个精子与约7.5pL50μg/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IVF7～10h时注射的分裂率、早期胚胎基因表达率均显著高于18～20h(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7～10h注射的效果优于IVF后18～20h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 oocytes 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 injection 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 injection group were significantly higher with injection at 7-10 h post IVF than that at 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.