摘要
建立体外合成mRNA高效转染人胚胎干细胞(h ESCs)的方法,为高效基因转染h ESCs提供一种新选择。体外合成绿色荧光蛋白mRNA(e GFP mRNA)通过脂质体和电转的方法转染h ESCs,流式检测阳性率,台盼蓝染色检测细胞存活率。HN4人胚胎干细胞中转染不同剂量的e GFP mRNA和e GFP质粒,流式检测阳性率和荧光强度。将e GFP mRNA转染HN4和i PS细胞系,流式检测其转染效率。免疫荧光检测电转e GFP mRNA对HN4细胞干性的影响。体外合成红色荧光蛋白(DsRed mRNA)和e GFP mRNA共转HN4细胞荧光显微镜观察共转效果。体外合成e GFP mRNA可通过电转的方式高效进入HN4细胞中,并且电转后细胞状态良好,细胞存活率为94.6%±0.67%,在h ESCs和i PS细胞系中转染效率均达到99%以上。HN4细胞中电转低剂量的e GFP mRNA即可高效进入细胞并持续高效表达4 d。电转e GFP mRNA的HN4细胞可保持良好的干细胞特性,并且多种体外合成mRNA可高效共转h ESCs。体外合成mRNA可通过电转的方式高效转入h ESCs中,体外合成mRNA技术为h ESCs高效基因转染提供了一个新思路。
Transfection of the in vitro synthesized mRNA into the human embryonic stem cells(h ESCs)with high efficiency will pave the way to gene manipulation of the h ESCs.In this study the authors synthesized the e GFP mRNA in vitro and transfection it into the h ESCs by different methods and the transfection efficiency was detected by flow cytometry.The results showed that the e GFP mRNA could transfect into the h ESCs by electroporation with the efficiency of more than 99%while the cells transfected by the chemical transfection reagent with only 15%.To study the cell survival rate after electroporation,the authors stained the cells with Taipan Blue and count the cells under the microscope.By counting and calculating the survival rate of cells after electroporation was94.6%±0.67%.To make sure whether electroporation would influence the pluripotency of h ESCs or not,the authors detected the pluripotency markers of the embryonic stem cells after electroporation by immunofluorescence.The results showed that after electroporation the e GFP mRNA into the h ESCs of the cells expression,the key markers of pluripotency and this transfection method didn’t influence the pluripotency of h ESCs.Electroporation of the i PS cells also could get the high efficient with the efficiency of more than99%.To compare the transfection efficiency and the duration of DNA and mRNA into the h ESCs,the e GFP mRNA or e GFP plasmid with different doses were transfected into the h ESCs and the fluorescence intensity was detected by flow cytometry.The e GFP mRNA could enter the h ESCs with low dosage and expression in the cells for four days.The co-transfection efficiency of e GFP mRNA and DsRed mRNA was observed by fluorescence microscope.The mRNAs could co-transfection into the h ESCs with high efficiency.The authors’study showed that in vitro synthesized mRNA could enter the h ESCs with high efficiency by electroporation.This will be a useful tool to increase the transfection efficiency of genes into the h ESCs and lay the foundation of using this technique induce the h ESCs differentiation into other cell types for clinical use.
作者
吴栋
马石楠
杨梦洁
王小莉
WU Dong;MA Shi-nan;YANG Meng-jie;WANG Xiao-li(Key Laboratory of Embryonic Stem Cell Research of Hubei Province,Department of Taihe Hospital,Hubei University of Medicine,Shiyan 442000,China)
出处
《药物生物技术》
CAS
2019年第6期477-480,共4页
Pharmaceutical Biotechnology
基金
湖北省科技重大专项(No.2016ACA157)
湖北医药学院博士启动金(No.2018QDJZR02)
湖北医药学院国家级创新训练项目(No.201610929009)
国家级创新训练项目(No.201710929010)。
关键词
体外合成mRNA
电转
人胚胎干细胞
基因转染
高效
共转
In vitro synthesized mRNA
Electroporation
h ESCs
Gene transfection
High efficiency
Co-transfection
