应用CRISPR/Cas9慢病毒系统建立Nestin基因敲除的小鼠肾足细胞系

摘要

Objective To establish mouse podocytes Nestin gene knockout cell lines stably based on CRISPR/Cas9 system.Methods Using Cas9 overexpression of lentivirus to build Cas9 overexpression of conditional permanent resident mouse podocytes cell lines.Three gene loci of Nestin (KO1、KO2、KO3)and three pairs of sgRNAs targeting Nestin were designed and inserted in GV371 vector to construct GV371-Nes-sgRNA expression vector for knockout.After sequenced and packed lentivirus particles,then used them to transfect the built CAS9 overexpress MPC.Then puromycin was used to screen positive cells.The enzyme of Cruiser TM to verify CAS9- sgRNA' shear of targeting gene activity.Results Cas9/MP and recombinant plasmid of GV371-Nes-sgRNA was constructed successfully.Cruiser TM verified that the knockout activity of CAS9-sgRNA2 was the maximum. Conclusion The stable MP cell lines of Nestin-knockout are successfully generated by CRISPR/Cas9 system,which provides the foundation for further studying the functions of Nestin gene.%目的 基于CRISPR/Cas9系统构建稳定敲除Nestin基因的条件永生性小鼠肾足细胞株(mouse podocyte,MP).方法 利用Cas9过表达慢病毒构建条件性永生小鼠足细胞Cas9过表达稳转细胞系(CAS9/MP).设计Nestin基因的3个位点(KO1、KO2、KO3),并构建3对靶向Nestin基因的sgRNA寡核苷酸序列,将其连接进GV371质粒中,测序验证后,包装慢病毒颗粒,感染构建好的CAS9/MP,嘌呤霉素筛选(Puromycin)阳性克隆细胞,Cruiser TM酶切检测CAS9-sgRNA对目的基因的剪切活性.结果 成功构建了CAS9/MP及GV371-Nes-sgRNA重组载体,CruiserTM酶法验证表明,CAS9-sgRNA2具有剪切活性.结论 建立能稳定敲除Nestin基因的CRISPR/Cas9慢病毒系统,成功获得Nestin敲除的MP稳转细胞株.为进一步研究Nestin在足细胞中的作用机制奠定基础.