潮霉素抗性3T3细胞的建立和鉴定

目的　建立具有潮霉素 (hygromycin)抗性的 3T3细胞系 ,用于转染目的基因 (pTRE Ins human)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 ,将含有潮霉素B磷酸转移酶基因的质粒pHyg导入 3T3细胞中 ,利用潮霉素的药物选择特性 ,对转染细胞进行压力筛选 ,并对其进行PCR鉴定。结果　经 5 0 0 μg ml的潮霉素压力筛选后 ,获得了抗性细胞克隆。抗性 3T3细胞的形态和生长速度与正常 3T3细胞没有差异 ,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。结论　成功地培育了潮霉素抗性的 3T3细胞 ,为进行目的基因 (pTRE Ins human)转染ES细胞的阳性细胞克隆筛选奠定了基础。

Development and Identification of a Hygromycin-Resistant Feeder Layer of ES Cell of Mouse

Objective In order to obtain a feeder layer for ES cells transfected by pTRE-Ins-human gene by means of developmed of the hygromycin-resistant 3T3 cell line. Methods The plasmid pHyg containing hygromycin gene was purified and transfected into 3T3 cells with Lipofectin.The transfected cells would be survived in further culture medium containing hygromycin B antibiotic as the hygromycin gene could express a hygromycin resistant products.The identification of the hygromycin resistant 3T3 cell line was conducted by polymerase chain reaction with the specific primers of hygromycin gene. Results The hygromycin R 3T3 cell line was established successfully.There were no differences among hygromycin R 3T3 cells and normal 3T3 cells morphologically and propagatively. The hyg gene DNA fragment could be amplified by PCR with specific primers in the genomic DNA of hygromycin R 3T3 cells. Conclusion The hygromycin R 3T3 cells were established with transfection of plasmid pHyg by Lipfectin,which may be useful for selecting ES cells transfected by pTRE-Ins-human gene.