电穿孔介导小干扰RNA高效转染小鼠附植前胚胎

摘要

使用Cy3标记的阴性对照小干扰RNA (siRNA)转染小鼠附植前胚胎,建立向小鼠附植前胚胎导入siRNA的电穿孔方法.通过控制透明带弱化程度、电压、脉冲时间和脉冲次数等条件,采用不同参数组合并结合使用不同介质作为电转缓冲液将Cy3标记的阴性对照siRNA转染小鼠附植前胚胎.在荧光倒置显微镜下,观察胚胎的存活率、siRNA转染率以及阳性转染存活胚胎的囊胚发育率.结果显示小鼠附植前胚胎在使用台氏液消化胚胎透明带10s后,以opti-MEM作为电转缓冲液,电穿孔参数设置为30 V,1 ms,3次的条件下取得最佳转染效果.总之,电穿孔方法可实现siRNA简便、高效地转染小鼠附植前胚胎.%We developed a detailed electroporation method to deliver efficiently siRNA into mouse preimplantation embryos. By introducing Cy3 labeled negative control small interfering RNA (siRNA) into mouse preimplantation embryos, we optimized conditions for the electroporation, including the voltage, pulse duration, pulse number, electroporation buffer and an important step to weaken the zona pellucida. Embryonic survival rate, transfection rate and blastocyst development rate were evaluated under the converted fluorescence microscope, by embryos counting and statistical analysis. The best transfection was achieved in opti-MEM under the conditions of 30 V, 1 ms, 3 pulses, and the duration of digestion in tyrode's solution was 10 s. In conclusion, the proposed electroporation approach here is a simple and efficient tool to deliver siRNA for RNA interference (RNAi) into mouse preimplantation embryos.