CRISPR/Cas9介导下UPS系统-E3泛素连接酶ITCH基因敲除细胞株的初步构建

Preliminary Construction of UPS-E3 Ubiquitin Ligase ITCH Gene Knockout Cells Mediated by CRISPR/Cas9

目的:构建CRISPR/Cas9介导下泛素-蛋白酶体系统(ubiquitin proteasome system,UPS)-E3泛素连接酶(atrophin 1interacting protein 4,ITCH)基因敲除细胞株。方法:在人ITCH基因的PAS结构域内设计3个sgRNA,构建3个分别同时表达ITCH-sgRNA、Cas9和eGFP的三合一质粒。经测序序列正确的重组质粒用lipofectamine 3000转染HeLa细胞,24h后荧光倒置显微镜观察转染情况。结果:3个ITCHsgRNA寡核苷酸单链成功退火成为双链,经测序发现,设计的3个ITCH-sgRNA全部重组到了CRISPR/Cas9载体上。荧光检测发现在同时表达ITCH-sgRNA、Cas9和eGFP的三合一质粒转染的HeLa细胞内有绿色荧光。结论:成功构建了同时表达ITCH-sgRNA、Cas9和eGFP的三合一质粒,并将三合一重组质粒成功转入细胞中,为实现在HeLa细胞中完全敲除ITCH基因,从而为构建ITCH基因敲除稳定株奠定了基础。

Objective:To constructionofUPS-E3ubiquitinligaseITCH geneKnockout Cells mediated by CRISPR/Cas9.Methods:Three sgRNAs in the PAS domain of the human ITCH gene were designed,constructed of 3 three-in-one plasmids that simultaneously expresses ITCH-sgRNA,Cas9 and eGFP.Recombinant plasmids with correct sequencing sequence were transfected into HeLa cells using Lipofectamine 3000,after 24 hours,inspected the transfection status under a fluorescence inverted microscope.Results:Three ITCH-sgRNAs oligonucleotide single strands were successfully annealed into double strands,after sequen-cing,it was found that three ITCH-sgRNAs were recombined onto the CRISPR/Cas9 vector.Fluorometric assay showed the green fluorescence was observed in HeLa cells transfected with recombinant plasmids.Conclusion:Successfully constructed three-in-one plasmids that simultaneously expresses ITCH-sgRNA,Cas9 and eGFP,the recombinant plasmids are successfully transfected into HeLa cells,to achieve complete knockout of the ITCH gene in HeLa cells,thus laying the foundation for constructing stable ITCH gene knockout strains.