电穿孔介导外源基因转染兔成体成纤维细胞的初步研究

›› 2010, Vol. 30 ›› Issue (4): 241-245.

电穿孔介导外源基因转染兔成体成纤维细胞的初步研究

1.上海交通大学医学院实验动物科学部，上海，200025；2.东北林业大学野生动物资源学院，哈尔滨，150040；3.新疆农业大学动物医学学院，乌鲁木齐，830052

收稿日期:2009-12-10 出版日期:2010-04-30 发布日期:2010-04-30
基金资助:
国家自然科学基金（30770324）；国家转基因重大专项（No.2009ZX8006-014B）；上海市科委科研计划项目（0914091000）；上海市农委科技兴农重点攻关项目（农科攻（2006）字5-3号）（农科攻（2005）第3-5号）

Preliminary Study on Conditions of Transfection of Rabbit Adult Fibroblasts by Electroporation

1.Department of Laboratory A nimal Science, School ofMedicin, Shanghai JiaoTong University Shanghai 200025, China; 2. College of Wildlife Resoure of Northeast Forestry University, Harbin 120040，China;3 College of A nimal Medicine ofXinjiang Agricultural University, Wulumuqi 830052, China

Received:2009-12-10 Online:2010-04-30 Published:2010-04-30

摘要/Abstract

摘要： 目的采用电穿孔方法，介导质粒载体pEGFP-c1转入兔成体成纤维细胞，探讨建立电穿孔介导外源基因转染兔成体成纤维细胞的基本条件。方法电穿孔介导质粒载体pEGFP-c1转入7-10代兔耳成体成纤维细胞系GG，研究不同电压、电容、DNA剂量，孵育温度的条件下，观察细胞存活数，流式细胞仪测试GFP的含量。结果以PBS作为电击基础液、细胞浓度2×10⁶个/ml，电压250 V、电容700 μF，DNA用量30 μg时，兔耳成纤维细胞的转染率和存活率最佳。且电击前冰浴10 min，电转染后37℃孵育10 min有利于提高细胞的转染率和存活率。结论在合适电压、电容、DNA剂量条件下，电转染提供了外源基因转染兔耳成体成纤维细胞适用性；电转染前的一定时间冰浴和电转染后一定温度孵育可使电转染效率得到改善。

关键词: 电穿孔, 兔成体成纤维细胞, pEGFP-c1, 转染

Abstract: Objective To investigate and explore conditions of tansfection on the rabbit adult fibroblasts by means of electroporation, the plasmid vector of pEGFP-cl was successfully transfected into the rabbit adult fibroblasts by electroporation. Methods The rabbit fibroblasts at 7-10 passages were transferred with pEGFP-cl by means of electroporation with different electric voltages, capacitors，the doses of DNA, different incubation time before electroporation, incubation temperature after electroporation. The cell survival rate was counted under visible light microscope, and the transfection efficiency and production of protein were identified through FACS analysis. Results transfection efficiency and cell survival rate were of the higher value, when The rabbit fibroblast cell with concentra-tion of 2 × 10⁶cells/ml (electroporation medium: PBS) and mixed with 30 jig vector DNA，the electric voltage of electropration apparatus was set at 250 V, and the capacitors was 700 jiiF. Moreover，the electransfected efficiency and cell survival rate was increased with 10 min of ice incubation before electroporation and 10 min of 37℃ incubation after electroporation. Conclusion The electrotransfection is very critical for the tansfection on the rabbit adult fibroblasts，and incubation temperature could improve transfection efficiency.

Key words: Electroperation, Rabbit adult fibroblast cell, pEGFP-cl, Transfection

谷瑞环1，李善刚1，宋伟杰2，王伟3，丁雷2，张艳1，邢凤英1，李垚1，陈学进1. 电穿孔介导外源基因转染兔成体成纤维细胞的初步研究[J]. , 2010, 30(4): 241-245.

GU Rui-huan1，LI Shan-gang1，SONG Wei-jie2，WANG Wei3，DING Lei2，ZHANG Yan1，XING Feng-ying1，LI Yao1，CHEN Xue-jin1. Preliminary Study on Conditions of Transfection of Rabbit Adult Fibroblasts by Electroporation[J]. , 2010, 30(4): 241-245.

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