精子介导GFP基因在小鼠早期胚胎中的表达

Expression of GFP Gene in Mouse Early Embryo Produced by Sperm-mediated Gene Transfer

利用DIG末端标记技术和免疫组化技术分析了小鼠精子体外结合内化外源DNA的效率。试验结果表明,不同小鼠个体的精子结合外源DNA的阳性率有明显差异(P<0.01),平均为13%。利用考马斯亮蓝染色评价了小鼠精子顶体反应发生的情况,筛选出TYH培养液为较合适的体外受精液。利用小鼠体外受精技术,将体外转染GFP基因并获能的小鼠精子与成熟卵母细胞进行体外受精,受精卵进行体外培养,表达GFP胚胎的阳性率为4.7%。验证了精子介导制备转基因小鼠胚胎的可行性,并建立了利用精子载体法制备转基因小鼠胚胎的平台。

The efficiency of the exogenous DNA transfecting mouse sperm was studied by the DIG end labeled and immunohistochemistry technology. The results suggested that： the efficiency of transfecting positive rate of individual mouse sperm was distinct difference （P 〈 0.01）, and the average rate was 13%. The acrosomal reaction was evaluated using the technology of Coomassie brilliant blue stained, and the appropriate in vitro fertilization （IVF） medium TYH was elected. Mouse sperms were transferred with GFP gene in vitro, and the mature oocytes were fertilized using IVF, and then the zygotes were cultured in vitro. The embryos were observed using the fluorescence microscopy, and the transgenic rate was 4.7%. The results suggested that sperm mediated gene transfer （SMGT） was an effective and feasible method.