抗G418小鼠胚胎干细胞饲养层的制备

Development of a G418-resistant feeder layer of embryonic stem cell of mice

目的:建立具有G418抗性的 3T3细胞系,用于转染pTet on基因的ES阳性细胞克隆筛选的饲养层。方法: 通过脂质体转染的方法,将含有neo基因的质粒pWL/neo导入 3T3细胞中,利用G418的药物选择特性,对转染细胞进行压力筛选,并对其进行PCR鉴定。结果:经 500μg/ml的G418压力筛选后,获得了抗G418细胞克隆。抗性 3T3细胞的形态和生长速度与正常 3T3细胞没有差异,用特异性核苷酸引物检测抗性细胞基因组DNA,可以扩增出对应的核苷酸片段,Southernblot鉴定结果表明neo基因片段已整合入G418抗性 3T3细胞中。结论:成功地培育了G418抗性的 3T3细胞,为进行pTet on基因转染ES细胞的阳性细胞克隆筛选奠定了基础。

Objective: In order to obtain a feeder layer of embryonic stem (ES) cells transfected by pTet-on gene by means of the G418-resistant 3T3 cell line established. Methods: The plasmid pWL/neo containing neo gene was purified and transfected into 3T3 cells with Lipofectin.The transfected cells would be survived in culture medium containing G418 antibiotic because the neo gene could express a G418 resistant product.The identification of the G418-resistant 3T3 cell line was conducted by using polymerase chain reaction (PCR) with the specific primers of neo gene. Results: Under the condition of G418 (500 μg/ml ), G418-resistant 3T3 cell clones have been successfully selected. The G418-resistant 3T3 cells had no difference from normal 3T3 cells in morphology and propagation rate. The neo gene DNA fragment could be amplified by using PCR with specific primers in the genomic DNA of G418-resistant 3T3 cells. Southern blot analysis showed that neo fragment was integrated into G418-resistant 3T3 cells. Conclusion: The G418-resistant 3T3 cells were established successfully by Lipfectin,which may be useful of selecting ES cells transfected by pTet-on gene.