【摘要】 [目的]建立具有潮霉素B(hygromycinB)抗性的3T3细胞系,用于转染目的基因(pTRE2-human-Ins)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(hyg)的质粒pHyg导入NIH3T3细胞中,利用潮霉素B的药物选择特性,对转染细胞进行压力筛选,并对其进行PCR和southernblot鉴定。[结果]经300ug/ml的潮霉素B压力筛选后,获得了抗性细胞克隆。抗性NIH3T3细胞的形态和生长速度与正常NIH3T3细胞没有差异,特异性核苷酸引物检测抗性细胞基因组DNA,可以扩增出相应的核苷酸片段,Southernblot鉴定结果表明潮霉素基因片段已整合入潮霉素抗性NIH3T3细胞。[结论]本实验通过脂质体介导的方法成功地培育了潮霉素B抗性的NIH3T3细胞,为进行目的基因(pTRE2-human-Ins)转染ES细胞的阳性细胞克隆筛选打下了基础。更多还原

【Abstract】 [Objective] In order to obtain a feeder layer of ES cells transfected with pTRE-Ins-human gene, a hygromycin B resistant NIH3T3 cells were established.[Methods] the plasmid pHyg containing hygromycin phosphofransferose gene was purified and transfected into NIH3T3 cells with Lipofectin. The transfected cells were selected with the culture medium containing hygromycin B. The identification of the hygromycin B resistant NIH3T3 cell line was identified by polymerase chain reaction with the specific primers of hygromycin phosphofransferose gene. [Reults] NIH3T3 cell clones were successfully selected in the culture medium containing 300μg/ml hygromycin B. The hygromycin B resistant NIHNIH3T3 cells were no differences from normal NIH3T3 cells in morphology and propagation rate. The hyg phosphofransferose gene DNA fragment could be amplified by PCR with specific primers in the genomic DNA of hygromycin B resistant NIH3T3 cells; Southern blot analysis showed that hygromycin phosphofransferose fragment was integrated into NIHNIH3T3 cells. [Conclusion] The hygromycin B resistant NIH3T3 cells were established which may be useful for selecting ES cells transfected by pTRE-Ins-human gene.更多还原