畜牧兽医学报2012，Vol.43Issue(1)：159-166,8.
山羊乳腺上皮细胞基因转染条件的优化
Optimization of Electroporation for Transfection of Goat Mammary Epithelial Cells
摘要
Abstract
This study was conducted to obtain the optimal electroporation parameters for transfec-tion of goat mammary epithelial cells. Mammary epithelial cells of diary goat were cultivated successfully using tissue mass inoculation method, which were identified by immunofluorescence staining of cytokeratin 18 and detecting the growth curve. And then the effectiveness of different voltage (120, 140, 160, 180, 200 V), different pulse length (5, 10, 15, 20 ms), and two different cell lines (GMEC1, GMEC2) of the first generation, 3rd generation, 5th generation, 9th generation were compared. Finally, it was found that one 15 ms pulses of 180 V can introduced DNA into the cells effectively. The results inferred that passage numbers exert more remarkable influence on transfection efficiency than genetic background. The low-passage mammary epithelial cells had the highest transfection efficiency. The ability of these electroporation parameters to produce stably transfected goat mammary epithelial cells was determined following G418 selection with transfecting another vector, the goat mammary epithelial cell clonies was got, which express the exogenous human-beta-defensin-3 gene stably. This study provides the optimal electroporation parameters for introducing DNA into mammary epithelial cells and improves the efficiency of transgenic cloning by SCNT.关键词
山羊乳腺上皮细胞/电穿孔/转染效率/人β-防御素3基因/诱导表达Key words
goat mammary epithelial cells s electroporation/ transfection efficiency/ human beta defensin 3 gene/ induced expression分类
农业科技引用本文复制引用
杜珊,马保华,李亮亮,刘军,赵晓娥,张涌..山羊乳腺上皮细胞基因转染条件的优化[J].畜牧兽医学报,2012,43(1):159-166,8.基金项目
国家转基因生物新品种培育重大专项(2009ZX08008-010B) （2009ZX08008-010B）
