猪胎儿成纤维细胞的分离与基因转染

In vitro Culture of Porcine Fetal Fibroblast Cells and Gene Transfection

为利用核移植法获取转基因克隆猪提供供体细胞,体外分离培养猪胎儿成纤维细胞并进行了绿色荧光蛋白基因转染。首先采用组织块贴壁法分离培养猪胎儿成纤维细胞,绿色荧光蛋白表达载体以脂质法介导转染猪胎儿成纤维细胞,通过G418筛选获得稳定转染的细胞克隆,PCR鉴定外源基因在细胞基因组中的整合,利用荧光显微镜检测荧光蛋白的表达。结果表明,组织块贴壁后9 d可获取原代成纤维细胞,基因转染后利用G418筛选9 d即可获得转基因细胞,对转基因细胞进行传代,PCR检测显示荧光蛋白基因整合到细胞基因组中,荧光显微镜下观察到绿色荧光蛋白表达,为下一步通过核移植方法获得转基因猪提供了基础。

In order to offer donor cells for porcine transgenic cloning by nuclear transferring, fibroblast cells were isolated by attaching tissue explants from porcine fetal. The plasmid containing green-fluorescent protein cDNA was transfected into porcine fetal fibroblast by Lipofectmine. Cell clones were obtained after screening by G418. The recombinant of extrogenous DNA was identified by polymerase chain reaction, the extrogenous DNA expression was identified by fluorescence microscope. Results showed that primary porcine fibroblast cells were isolated from the tissue cultured for 9 days, and the transgenic cells were obtained after G418 selection for 9 days. Identification of the transgene in the cell clones was examined by PCR and the exogenous DNA had been integrated into genome. The transgenic cell line could express green-fluorescent protein. These results have paved the way to obtain the new transgenic porcine by nuclear transfer in the future.