电穿孔导入绿色荧光蛋白基因于绵羊胎儿成纤维细胞研究

本文应用质粒pEGFP Cl带有水母绿色荧光蛋白 (GFP)基因和新霉素抗性基因 (Neor)两种报告基因转染胎儿成纤维细胞 ,进行转染条件优化。首先 ,通过成活率确定转染脉冲时程和场强 ,发现有三种组合是最适的组合 ,它们分别为 :时程 5ms,场强 1.2 5～ 1.5kV/cm ;时程 15ms时 ,场强 1.0～ 1.2 5kV/cm ;时程 2 5ms时 ,场强 0 .75～ 1.0kV/cm。接着采用场强 1.2 5kV/cm ,时程 15ms ,研究转染的温度、电导介质、细胞状态、DNA用量和细胞密度对转染效率的影响 ,结果表明电导介质为Dhanks,DNA用量为 4 μg/mL ,对数期细胞密度为 1× 10 6～ 1× 10 7个 /mL ,电击前后在 4℃下各静置 10min时 ,能获得最佳转染效率。

Studies on Introduction of GFP Gene into Sheep Fetal Fibroblast Cell by Electroporation

In the study of optimizing transfection procedure, the plasmid pEGFP-Cl(with GFP and Neo~r as reporter gene) was transfered into sheep fetal fibroblast cells by electroporation. First, three electric fields were identified as the best according to the surviving rate of cells, of which the electric strength and pulse duration are 1.25～1.5 kV/cm for 5 ms, 1.0～1.25 kV/cm for 15 ms and 0.75～1.0 kV/cm for 25 ms respectively. Afterwards, the electric field was stabilized at 1.25 kV/cm and with a pulse duration of 15 ms to study the factors such as temperature, plasmid DNA concentration, number and phase of cells as well as conducting medium that affect the transfecting efficiency. The results showed that, under the condition of Dhanks as conducting medium, 4 μg/mL plasmid DNA, 1×10~6～1×10~7doubling cells/mL and holding the fluid at 4℃ for ten minutes before and after transfection , the efficiency was proved to be the highest one.