组织工程与重建外科杂志

组织工程与重建外科杂志 ›› 2019, Vol. 15 ›› Issue (4): 215-221.doi: 10.3969/j.issn.1673-0364.2019.04.001

• 论著 •    下一篇

电转方法用于modRNA转染细胞的初步研究

陈茂林，王会景，艾雪峰，颜冰倩，宫艺奇，谭瑶，付炜，王伟   

  1. 上海交通大学医学院附属上海儿童医学中心心胸外科；上海交通大学医学院附属上海儿童医学中心转化医学研究所；上海市组织工程研究重点实验室
  • 收稿日期:2019-06-17 修回日期:2019-07-20 出版日期:2019-08-20 发布日期:2019-08-20
  • 作者简介:付炜(E-mail:fuweizhulu@163.com);王伟(E-mail:wangweicpb@yahoo.com);

Preliminary Study of Electroporation Method for modRNA Transfected Cells

CHEN Maolin,WANG Huijing,AI Xuefeng,YAN Bingqian,GONG Yiqi,TAN Yao,FU Wei,WANG Wei   

  • Received:2019-06-17 Revised:2019-07-20 Online:2019-08-20 Published:2019-08-20
  • Contact: 国家自然科学基金项目(81670372)；上海市浦江人才计划(18PJD031)；上海交通大学医学院转化医学协同创新中心合作研究项目(TM201821)；上海交通大学医工交叉项目(YG2016MS28)；上海市组织工程重点实验室开放课题

摘要： 目的探讨电转方法用于modRNA转染细胞的可行性,并筛选出不同细胞电转所需的最适电压和脉冲时间。方法 (1)筛选电转EGFP modRNA进入人成纤维细胞的最佳电压和脉冲时间参数;(2)在此基础上将人成纤维细胞转染n GFP/EGFP+mCherry modRNA;(3)将Hela、293T、3T3三种常见贴壁细胞电转EGFP modRNA。结果 (1)人成纤维细胞电转modRNA的最适电压为440 V,最适脉冲时间为30 ms;(2)nGFP可在核内表达,EGFP和mCherry两种蛋白可同时在胞质中表达;(3)Hela、293T、3T3细胞电转最适电压分别为425 V、400 V和440 V,最适脉冲时间均为30 ms。结论本实验证明(1)电转法转染modRNA进入细胞质和细胞核的可行性;(2)可同时将两种modRNA导入同一个细胞内并表达;(3)最适电压和最适脉冲时间需根据不同的细胞种类分别筛选。

关键词: 合成修饰的信使核糖核酸, 电转法, 最适电压, 最适脉冲时间

Abstract: Objective To investigate the feasibility of electroporation for modRNA transfection and to screen the optimal voltage and pulse time required for different cell electroporation. Methods ①The optimal voltage and pulse time parameters for electroporation of EGFP modRNA into human fibroblasts were screened;②Human fibroblasts were transfected with nGFP and EGFP+mCherry modRNA;③Hela, 293T, 3T3, three common adherent cells were electroporated with EGFP modRNA. Results ①The optimum voltage for electroporation of human fibroblasts was 440 V, and the optimal pulse time was 30 ms;②nGFP could be expressed in the nucleus;EGFP and mCherry proteins could be expressed simultaneously in the cytoplasm;③The optimum voltages for Hela, 293T and 3T3 cells were 425 V, 400 V and 440 V respectively, and the optimal pulse time was 30 ms. Conclusion This experiment demonstrates:①The feasibility of transfection of modRNA into cytoplasm and nucleus by electroporation;②Simultaneous introduction of two modRNAs into the same cell and expression;③ Optimal voltage and optimal pulse time are based on different cell types.

Key words: modRNA, Electroporation, Optimal voltage, Optimal pulse time

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