应用CRISPR-Cas9技术构建RIG-Ⅰ基因敲除的HEK293细胞系  

Knockout of RIG-I in HEK293 cells by CRISPR/Cas9

作　　者：陈姿亦 吴怡蓉 张雨婷 高有领[1] CHEN Ziyi;WU Yirong;ZHANG Yuting;GAO Youling(College of Biological and Environmental Sciences,Zhejiang Wanli University,Ningbo 315100,Zhejiang,China)

机构地区：[1]浙江万里学院生物与环境学院,浙江宁波315100

出　　处：《生物工程学报》2024年第11期4254-4265,共12页Chinese Journal of Biotechnology

基　　金：浙江省基础公益研究计划(LGN22C190028)。

摘　　要：为了揭示维甲酸诱导基因Ⅰ(retinoic acid inducible-geneⅠ,RIG-Ⅰ)基因敲除对Ⅰ型干扰素信号通路中关键因子的影响,本研究利用CRISPR/Cas9技术构建了RIG-Ⅰ基因敲除的HEK293细胞。首先设计了3条针对靶标基因的单向导RNA(single guide RNA,sgRNA),并构建了pX459重组载体。重组载体转染HEK293细胞后,通过嘌呤霉素筛选细胞株,再以聚肌苷酸-聚胞苷酸(polyinosinic acid-polycytidylicacid,poly I:C)为病毒模拟物转染细胞,通过基因测序、荧光定量PCR、免疫印迹和免疫荧光的方法检测RIG-Ⅰ基因的敲除情况,同时对Ⅰ型干扰素信号通路中的黑色素瘤分化相关基因5(melanoma differentiation-associated protein 5,MDA5)、干扰素β1(interferonβ1,IFNβ1)和核转录因子kappa B p65[nuclear transcription factor kappa B p65,NF-κb(p65)]等关键因子以及细胞活力进行分析。结果表明,本研究成功构建了2株稳定敲除RIG-Ⅰ基因的HEK293细胞株(S1和S3),RIG-Ⅰ在S1和S3中的基因转录水平和蛋白质表达水平均显著低于野生型细胞(P<0.05)。S1和S3细胞株的MDA5和IFNβ1基因转录水平和S3细胞株的NF-κB(p65)蛋白质水平显著低于野生细胞株(P<0.05);细胞免疫荧光分析结果表明,poly I:C转染细胞后,与野生型相比,S1细胞株核外存在较多的NF-κB(p65)蛋白。此外,polyI:C转染细胞48h后,显著降低了野生型和S1细胞株的活力(P<0.05),但是不影响S3细胞株的活力。综上所述,本实验通过CRISPR/Cas9系统成功构建了2株RIG-Ⅰ基因敲除的HEK293细胞,为进一步研究I型干扰素信号通路的机制提供了稳定的细胞模型。We knocked out the retinoic acid-inducible gene I(RIG-I)in HEK293 cells via CRISPR/Cas9 to reveal the effects of RIG-I knockout on the key factors in the type I interferon signaling pathway.Three single guide RNAs(sgRNAs)targeting RIG-I were designed,and the recombination vectors were constructed on the basis of the pX459 vector and used to transfect HEK293 cells,which were screened by puromycin subsequently.Furthermore,a mimic of virus,poly I:C,was used to transfect the cells screened out.RIG-I knockout was checked by sequencing,real-time quantitative PCR,Western blotting,and immunofluorescence assay.Meanwhile,the expression levels of key factors of type I interferon signaling pathway such as melanoma differentiation-associated gene 5(MDA5),interferonβ1(IFNβ1),and nuclear factor-kappa B p65[NF-κB(p65)],as well as cell viability,were determined.The results showed that two HEK293 cell lines(S1 and S3)with RIG-I knockout were obtained,which exhibited lower mRNA and protein levels of RIG-I than the wild type HEK293 cells(P<0.05).The mRNA levels of MDA5 and IFNβ1 in S1 and S3 cells and the protein level of NF-κB(p65)in S3 cells were lower than those in the wild type(P<0.05).More extranuclear NF-κB(p65)protein was detected in S1 cells than in the wild type after transfection with poly I:C.Plus,the wild-type and S1 cells transfected with poly I:C for 48 h showcased reduced viability(P<0.05),while S3 cells did not display the reduction in cell viability.In summary,the present study obtained two HEK293 cell lines with RIG-I knockout via CRISPR/Cas9,which provided a stable cell model for exploring the mechanism of type I interferon signaling pathway.

关 键 词：CRISPR/Cas9 维甲酸诱导基因Ⅰ(RIG-I) 基因敲除 HEK293细胞 干扰素 

分 类 号：Q78[生物学—分子生物学]