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目的:优化CUY21EDITⅡ电转仪介导真核表达载体p EGFP-N1转染大鼠来源的神经干细胞(NSCs)的转染条件,提高外源基因转染NSCs的转染效率,为后续试验提供基础。方法:在保持驱动电压(Pd V)、脉冲时间、循环次数等条件不变的情况下,通过改变电转电压(Pp V),将已构建好的表达绿色荧光蛋白的重组载体p EGFP-N1以CUY21EDITII电转仪转染导入NSCs,转染后48 h在荧光显微镜下观察荧光并计算转染效率。结果:p EGFP-N1质粒在Pp V为350 V,Pd V为20 V,脉冲时间为10 ms,循环次数为20次的转染条件下,可获得较理想的转染效率,转染效率平均为81.0%,细胞存活率为72.0%。结论:通过优化转染条件,CUY21EDITⅡ电转仪能提高p EGFP-N1转染NSCs的转染效率,与脂质体相比优势明显。
OBJECTIVE: To optimize the transfection conditions of transfected neural stem cells (NSCs) transfected with eukaryotic expression vector p EGFP-N1 by CUY21 EDIT Ⅱ electropositive apparatus and to improve the transfection efficiency of NSCs transfected with exogenous gene, so as to provide the basis for subsequent experiments. Methods: Recombinant vector p EGFP-N1 expressing green fluorescent protein (EGFP-N1) was constructed by changing the electropotentiation voltage (Pp V) under the condition of keeping the driving voltage (Pd V), pulse time, CUY21EDITII electrotransducer transfected into NSCs, 48 h after transfection fluorescence was observed under a fluorescence microscope and transfection efficiency was calculated. Results: The transfection efficiency of p EGFP-N1 plasmid under the conditions of 350 V Pp V, 20 V Pd V, 10 ms pulse time and 20 cycles was higher than that of the control Was 81.0%, cell survival rate was 72.0%. CONCLUSION: CUY21EDITⅡ electrotransducer can improve the transfection efficiency of p EGFP-N1 transfected NSCs by optimizing the transfection conditions, which has obvious advantages over liposomes.