基于CRISPR/Cas9技术构建稳定敲除TRIF基因的RAW264.7细胞株及对IFN-β的影响  

Construction of a stable TRIF gene knockout cell model in RAW264.7 cells using CRISPR/Cas9 technique

作　　者：陈为[1] 王燕 金连海[1] 常影[1] 董明鑫 刘文森 许娜[1] CHEN Wei;WANG Yan;JIN Lian-hai;CHANG Ying;DONG Ming-xin;LIU Wen-sen;XU Na(Jilin Medical University,Jilin 132013,China;Institue of Military Veterinary,Academy of Military Medical Sciences,Changchun 1301229,China)

机构地区：[1]吉林医药学院,吉林吉林市132013 [2]军事科学院军事医学研究院军事兽医研究所,吉林长春130122

出　　处：《中国兽医学报》2020年第1期66-71,共6页Chinese Journal of Veterinary Science

基　　金：国家重点研究计划资助项目(2017YFC1200900)

摘　　要：利用CRISPR/Cas9技术构建稳定敲除TRIF基因的RAW264.7细胞株。首先,制备Cas9表达慢病毒,感染RAW264.7细胞,通过Puromycin抗性进行初步筛选,PCR法进行鉴定。其次,设计3条特异性识别TRIF基因的sgRNA(sgTi1、sgTi2、sgTi3),将其转入稳定表达Cas9蛋白的RAW264.7细胞株,荧光显微镜观察转入效果,PCR和Western blot法验证基因敲除情况。最后,挑选基因敲除效果最佳细胞株,经TRIF激活剂诱导后,提取细胞总RNA,荧光定量PCR检测IFN-β表达水平。结果显示:感染后的RAW264.7-Cas9细胞Cas9基因扩增产物升高,说明成功获得稳定表达Cas9基因的RAW264.7细胞。含目的基因的慢病毒感染RAW264.7-Cas9细胞后,荧光显微镜下可明显观察到目的基因已成功导入;PCR结果显示,与对照组比较,分别导入sgTi1、sgTi2、sgTi3的3组RAW264.7细胞TRIF表达均受到抑制,Western blot进一步验证3组RAW264.7细胞TRIF蛋白表达均下降且sgTi1组下降最显著,说明TRIF基因敲除成功。经TRIF激活剂诱导,与RAW264.7-Cas9-NC细胞比较,敲除TRIF基因后RAW264.7细胞IFN-βmRNA水平受到明显抑制。结果表明:利用CRISPR/Cas9技术成功构建了稳定敲除TRIF基因的RAW264.7细胞株。This study aimed at establishing a TRIF knockout cell strain by CRISPER/Cas9 system.The three sgRNA(sgTi1,sgTi2,sgTi3)was transfected into RAW264.7 cells,which were confirmed to expressing Cas9 by Puromycin.The result of knockout was identified by fluorescence microscope,PCR and western blot.The best group of sgTRIF1-transfected cells were activated by TRIF to observe secretion of IFN-β.The results represented that the amplification of Cas9 raised in those selected RAW264.7 cells.Under fluorescence microscope,the target gene was successfully introduced.PCR and Western blot showed that the expression of TRIF was significantly decreased,and the most as sgTi1 group.RT-PCR showed that the IFN-β mRNA level of RAW264.7 cells was significantly inhibited after TRIF gene knockout.The results indicated that TRIF knockout RAW264.7 cell line were established successfully.

关 键 词：CRISPR/Cas9技术 TRIF基因 基因敲除 RAW264.7细胞 

分 类 号：S852.2[农业科学—基础兽医学]