水牛胎儿成纤维细胞电转染条件的优化

Optimization of Electroporation Condition for Buffalo Fetal Fibroblast Cells

高活力的转基因细胞的制备是转基因克隆动物研究的关键环节。为了高效制备转基因细胞,外源基因转入水牛胎儿成纤维细胞的条件需要优化。采用组织块贴壁法分离获得水牛胎儿成纤维细胞,经染色体核型分析表明获得的细胞具有正常的生物学功能,可用于细胞转染。采用电穿孔法将载体EGFP-N1导入水牛胎儿成纤维细胞,分别比较不同电压(200V,250V,300V,350V)、不同电击时间(5ms,10ms,15ms)和不同细胞代数(第3代、第5代、第10代)的转染效率,并利用优化的电转染条件将乳腺特异性表达的水牛促乳素(PRL)基因导入水牛胎儿成纤维细胞,G418筛选获单克隆并进行鉴定。结果发现电压为300 V,脉冲时间为10 ms时,细胞GFP阳性率为35.5%,转染效率较高。随传代次数的增加,细胞的转染效率下降;传至第10代的细胞转染效率显著低于第3代的水牛胎儿成纤维细胞(30%VS 35.5%,P<0.05)。运用优化条件转染、筛选可获得转PRL单克隆细胞,转染效率为2.1×10-5(84/4×106)。挑取单克隆细胞扩大培养,经PCR鉴定和核型分析,证实单克隆稳定整合促乳素基因,并且核型分析正常,可作为核供体,用于生产水牛转基因克隆胚胎。为采用体细胞克隆技术生产水牛转基因胚胎奠定了基础。

A significant aspect of applying somatic cell nuclear transfer （SCNT） technology to generate transgenic animal is the in vitro method utilized to obtain viable donor nuclei for cloning procedures. Therefore, the present study was undertaken to explore the optimal electroporation parameters for transfection of buffalo fetal fibroblasts （BFFs） and determine the ability of these electroporation parameters to produce stably transfected fibroblast colonies. First, the BFFs was obtained by tissue mass inoculation method and were confirmed of possessed a normal diploid karyotype. And then, the transfection efficiency of different voltage （150V, 200V, 250V, and 300V）, different pulse length （5ms, 10ms and 15ms） and different cells passage were compared.Results show that one 10ms pulse of 300V can introduced DNA into the cells more effectively, and the passage of cells also has remarkable influence on transfection efficiency. Finally, the utilization of these conditions can produced numerous transgenic fibroblast colonies following G418 selection. This study provides a suitable electroporation condition for introducing DNA into BFFs and improves the efficiency of transgenic SCNT technology.