摘要
该研究主要针对大质粒转染人源心室肌Ac16细胞的方式及最适条件进行摸索。采用人源心室肌Ac16细胞作为研究模型,分别用Lip3000转染、核酸脂质体(Hieff Trans;Liposomal)转染和慢病毒转染三种方法将外源大质粒pLenti-CasRx-EGFP导入Ac16细胞中,通过统计表达绿色荧光蛋白的阳性细胞比例,分析外源基因的转染效率,探索大质粒pLenti-CasRx-EGFP的最佳转染方式和转染条件。结果表明,Lip3000转染Ac16细胞的最适转染条件为:质粒与Lip3000的比例为1:2;核酸脂质体转染Ac16细胞的最适条件为:质粒与脂质体的比例为1:3;慢病毒感染Ac16细胞的最适MOI值为200;通过比较三种转染方式的转染效率发现慢病毒的转染效率最高。最后得出大于10 Kb的大型质粒(pLenti-CasRx-EGFP)转染人源心室肌Ac16细胞最适合的方式是利用慢病毒转染,最佳MOI值为200。
The aim of this study was to explore the transfection method and optimum condition of large plasmid transfecting human ventricular muscle Ac16 cell.The plasmid pLenti-CasRx-EGFP was transfected into Ac16 cells by three methods:Lip3000 transfection,Hieff Trans;Liposomal transfection and lentivirus transfection.The transfection efficiency of exogenous gene was determined by analyzing the proportion of positive cells expressing EGFP.Finally,the optimal transfection method and transfection condition of large plasmid pLenti-CasRx-EGFP was explored.For Ac16 cells,the optimum transfection ratio of plasmid to Lip3000 was 1:2;the optimal transfection ratio of plasmid to Hieff Trans;Liposomal transfection was 1:3;the optimal MOI value infected with lentivirus was 200.The transfection efficiency of lentivirus was the highest by comparing the efficiency of the three transfection methods.It is concluded that lentivirus is the most suitable method for transfection the large plasmid (pLenti-CasRx-EGFP) larger than 10 Kb into Ac16 cells with MOI of 200.
作者
李亚欢
范承昊
向晨莹
廉虹
LI Yahuan;FAN Chenghao;XIANG Chenying;LIAN Hong(Beijing Key Laboratory of Preclinical Research and Evaluation of Cardiovascular Implant Materials,Animal Experiment Center,Fuwai Hospital,National Center for Cardiovascular Diseases,Chinese Academy of Medical Sciences,Peking Union Medical College,Beijing 102300,China)
出处
《中国细胞生物学学报》
CAS
CSCD
2022年第2期299-305,共7页
Chinese Journal of Cell Biology
基金
国家自然科学基金(批准号:81900343)资助的课题。
