小鼠肌肉内绿色荧光蛋白基因电转染

Electrotransfer of EGFP gene-encoding plasmid into mouse muscles

目的 :将细胞电通透技术应用到 DNA肌肉转染并确定达到高效电转染的参数条件。方法 :以增强绿色荧光蛋白 (EGFP)基因质粒作为报告基因 ,进行小鼠体内肌肉组织电转染实验 ,在部分参数固定情况下 ,某一参数发生变化 ,通过荧光显微镜观察、公式计算得出转染率 ,进行比较。确定转染的最佳电场强度、脉冲时值和质粒剂量。结果 :EGFP质粒 15μg注射到小鼠的胫前肌 ,在注入后给予如下参数 ,即电脉冲 2 0 0 V· cm- 1 ,2 0 m s,1Hz,8次 ,转染率最高 ,可达 (73.2± 7.2 ) %。 EGFP质粒注射后不会在细胞外立即降解 ,注射质粒 DNA 1h内给予电脉冲不影响转染率 ;15μg为最适当的 DNA剂量 ;EGFP蛋白的表达 ,7d达到高峰。结论 :与单纯 EGFP质粒肌肉注射相比 ,DNA肌肉电转染可极大地提高其转染率。

Objective To delivery gene by electropermeabilization to skeletal muscles and choose the optimal parameters. Methods The enhanced green fluorescent proteins (EGFP) gene-encoding plasmid was used as reporter gene to investigate the conditions of best gene transfection with minimal muscle injury. Changing one parameter with other parameters fixed, transfection rates were compared,and optimal parameters to target plasmid DNA into skeletal muscles of mice were chosen. Results After 15 μg EGFP plasmid was injected into skeletal muscles of mice,8 square-ware pulses were applied at amplitudes 200 V·cm~ -1, with duration 20 ms and 1 Hz frequency,and the transfection rate was (73.2±7.2)%. Transfection efficiencies were same within 1 hour since it was stable.The optimal dosage of DNA was 15 μg. Maximum expression of EGFP fusion proteins was obstained 7 days after EGFP transfection. Conclusion DNA electrotransfection in muscle better than the only injection of EGFP plasmid into muscle.