鸡胚精原细胞体外转染EGFP方法的比较

Comparison on Transfection Methods of Chicken Spermatogonial Cells with Enhanced Green Fluorescent Protein in vitro

以增强型绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)为报告基因体外转染鸡胚胎精原细胞(Chicken embryonic spermatogonial cells,CESCs),比较磷酸钙、脂质体(梭华-Sofast)、电穿孔3种方法转染EGFP的效率,以获得高效的体外转染方法。本研究分离孵化16 d的鸡胚睾丸,获取CESCs,体外培养和传代扩增,传至第2代后进行碱性磷酸酶(AKP)活性和阶段特异性表面抗原-1(Stage specific embryonic antigen-1,SSEA-1)免疫荧光鉴定。运用磷酸钙、脂质体(梭华-Sofast)、电穿孔3种方法体外转染第2代CESCs,荧光细胞计数法分析转染效率。结果表明:与磷酸钙法、脂质体法相比,电穿孔法可获得更高的细胞成活率和转染率(68%vs39%、65%,19.70%vs2.92%、9.73%),差异显著(P<0.05)。因此得出,电穿孔法是将外源基因导入鸡胚精原细胞的适宜方法。

Enhanced green fluorescent protein （EGFP） gene was transfected into chicken embryonic spermatogonial cells （CESCs） in vitro by three different methods, calcium acid phosphate, liposome（Sofast）and electroporation, to obtain the optimal transfection strategy by detecting the transfection efficiencies. In this study, CESCs were isolated from chicken embryo testis on 16^th hatching days, then cultured and subcultured in vitro. CESCs were identified by AKP activity and SSEA-1 immunofluorescence assay at the 2^nd generation. Three different methods, calcium acid phosphate, liposome （Sofast） and electroporation, were used to transfect pEGFP-N1 plasmid into CESCs. The transfection efficiencies were measured by cell counting under fluorescent microscopy. The results indicated that transfecting CESCs by electroporation had significant higher cells survival rate and tansfection efficiency than that by calcium acid phosphate and liposome （Sofast） （68%vs39% ,65% ;19.70% vs 2.92% ,9.73%）. Therefore, we concluded that electroporation is the optimal transfection method to transfect exogenous gene into chicken embryonic spermatogonial cells in vitro.