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目的:建立稳定过表达Olig2的小鼠胚胎干细胞(mESCs)系。方法:从含有Olig2的质粒中利用PCR技术克隆Olig2基因编码序列,酶切连接入GV218载体,形成Olig2-GV218重组载体。将构建好的Olig2-GV218载体与两种病毒辅助包装原件载体质粒共转染293T细胞,制备并浓缩慢病毒颗粒,侵染mESCs,嘌呤霉素筛选的阳性克隆传代培养10代后鉴定细胞内Olig2的表达。结果:PCR扩增得到含Olig2基因编码序列的特异性条带。重组的Olig2-GV218载体中Olig2编码序列与目标序列几乎一致。携带有Olig2-GV218载体的慢病毒能够正常感染mESCs,表达Olig2-GFP融合蛋白,并稳定遗传。结论:成功建立了稳定过表达Olig2的小鼠胚胎干细胞系。
Objective: To establish a mouse embryonic stem cell (mESCs) line that overexpresses Olig2. Methods: The Olig2 gene was cloned from plasmid containing Olig2 by PCR and ligated into GV218 vector to form Olig2-GV218 recombinant vector. The constructed Olig2-GV218 vector was cotransfected into 293T cells with two vector-assisted packaging vectors, and the lentiviral particles were prepared and concentrated. The mESCs were infected and the positive clones selected by puromycin were subcultured for 10 generations, then the intracellular Olig2 expression. Results: Specific bands containing the Olig2 gene coding sequence were obtained by PCR. Olig2-GV218 recombinant vector encoding Olig2 almost identical with the target sequence. The lentivirus carrying Olig2-GV218 vector was able to infect mESCs normally, expressed Olig2-GFP fusion protein, and stably inherited. Conclusion: The mouse embryonic stem cell line that overexpresses Olig2 was successfully established.