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

作　　者：杜珊[1] 马保华[1] 李亮亮[1] 刘军[1] 赵晓娥[1] 张涌[1] 

机构地区：[1]西北农林科技大学动物医学院农业部动物生物技术重点实验室,杨凌712100

出　　处：《畜牧兽医学报》2012年第1期159-166,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家转基因生物新品种培育重大专项(2009ZX08008-010B)

摘　　要：为了优化外源基因转入乳腺上皮细胞的条件,本研究采用组织块培养法,从山羊乳腺组织中分离并获得了纯化的乳腺上皮细胞,通过染色角蛋白和测定生长曲线对其进行了鉴定和检测。然后利用电穿孔法将PEGFPC1载体导入乳腺上皮细胞,分别比较了在不同电压(120、140、160、180、200V)、不同电击时长(5、10、15、20ms)以及2株细胞(GMEC1、GMEC2)的不同代数(第1代、第3代、第5代、第9代)条件下的转染效率,并利用优化条件对山羊乳腺上皮细胞进行人β-防御素3基因转染,经过G418筛选,最终获得单克隆细胞,同时对获得的单克隆阳性细胞进行体外诱导表达,并对诱导产物进行Western blotting鉴定。结果表明,不同遗传背景的细胞对转染效率的影响较小,而第1代培养的细胞转染效率要显著高于传代细胞,乳腺上皮细胞在电压180V、电击时长15ms、电击1次的条件下转染效率最高,优化条件下转染、筛选和扩增后获得稳定表达人β-防御素3蛋白的单克隆乳腺上皮细胞。研究工作为乳蛋白基因表达调控机制的研究及乳腺特异性表达载体的检测提供参考资料。This study was conducted to obtain the optimal electroporation parameters for transfection 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 

分 类 号：S827[农业科学—畜牧学] S813.3[农业科学—畜牧兽医]