Position: Home > Articles > Construction of DDX21 knockout gene stable strain using CRISPR/Cpf1 gene editing technology and identification of functions
Chinese Journal of Veterinary Science
2020
(2)
257-263
利用CRISPR/Cpf1技术构建HEK293细胞DDX21基因稳定敲除株及功能鉴定
作 者：
鲁国涛;王辉;曾为俊;邵玉乐;许曼;陈洪岩;孟庆文
单 位：
中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/黑龙江省实验动物与比较医学重点实验室
关键词：
DDX21基因;H9N2亚型禽流感病毒;CRISPR/Cpf1;HEK293细胞;基因敲除
摘 要：
利用CRISPR/Cpf1基因编辑技术构建DDX21基因稳定敲除的细胞株,并检测其生物学功能。设计、构建靶向DDX21基因向导RNA表达载体,与Cpf1表达载体共转染HEK293细胞,通过流式筛选、基因测序、Western blot和细胞增殖能力检测鉴定细胞株;荧光定量PCR检测病毒感染后模式识别受体(TLR-3、TLR-7、MDA-5)、Ⅰ型干扰素、IL-6以及抗病毒蛋白OAS mRNA水平表达情况。结果显示:成功筛选到2株DDX21基因敲除的细胞株,蛋白免疫印迹显示DDX21蛋白不表达;与野生型(wild type,WT)相比敲除株形成细胞单层的时间明显延长(P<0.01);H9N2亚型禽流感病毒感染试验表明,模式识别受体TLR-3、MDA-5的mRNA表达水平上调,TLR-7表达水平无明显差异,IFN-α、IFN-β、IL-6及抗病毒蛋白OAS的mRNA表达水平下调,表明DDX21基因敲除阻断了DDX21-TRIF-MyD88信号通路;TCID_(50)测定结果显示,差异最高可达到WT的3.01倍,表明DDX21基因敲除后流感病毒复制增加。本研究利用CRISPR/Cpf1系统获得2株DDX21基因稳定敲除的HEK293细胞株,为深入研究DDX21基因功能、病毒感染的天然免疫应答和调控研究奠定理论基础。
译 名：
Construction of DDX21 knockout gene stable strain using CRISPR/Cpf1 gene editing technology and identification of functions
作 者：
LU Guo-tao;WANG Hui;ZENG Wei-jun;SHAO Yu-le;XU Man;CHEN Hong-yan;MENG Qing-wen;State Key Laboratory of Veterinary Biotechnology/Heilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences;
关键词：
DDX21 gene;;H9N2 subtype avian influenza virus;;CRISPR/Cpf1;;HEK293 cell;;gene knockout
摘 要：
To knock out DDX21 gene of HEK293 cells using CRISPR/Cpf1 gene editing technology and explore the biological functions of DDX21 knockout cell lines.The DDX21 gene-directed RNA expression vector was designed and constructed,co-transfected with Cpf1 expression vector into HEK293 cells.The cell lines were identified by flow cytometry,gene sequencing,Western blot and cell proliferation capacity.The expression level of pattern recognition receptor(TLR-3,TLR-7,MDA-5),type Ⅰ interferon,IL-6 and antiviral protein OAS mRNA were detected by real-time PCR after virus infection.The results showed that two cell lines without DDX21 were successfully obtained,and Western blot showed that DDX21 protein was not expressed.Compared with wild type(WT),the time to form cell monolayer was significantly prolonged(P<0.01).The experiment of virus infection by H9 N2 subtype avian influenza showed that the mRNA expression levels of pattern recognition receptors TLR-3 and MDA-5 were up-regulated significantly.However,there was no difference in TLR-7 expression levels,IFN-α,IFN-β,IL-6 and anti-virus.The mRNA expression level of OAS was down-regulated,indicating that DDX21 knockout blocked the DDX21-TRIF-MyD88 signaling pathway;the TCID50 assay showed that the difference was up to 3.01 times that of WT,indicating that the replication of influenza virus was increased after DDX21 knockout.In this study,two strains of DDK21 gene-stable HEK293 cell line were obtained by CRISPR/Cpf1 system,which laid a foundation for further study of DDX21 gene function,natural immune response and regulation of viral infection.
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