CRISPR/Cas9技术敲除RAW264.7细胞的GPR43基因抑制其对肺炎克雷伯菌的吞噬功能

CRISPR/Cas9-based knockout of GPR43 gene in RAW264.7 cells inhibits their phagocytosis to Klebsiella pneumoniae

目的利用成簇的规律间隔的短回文重复序列/Cas9核酸酶(CRISPR/Cas9)技术构建稳定敲除G蛋白偶联受体43(GPR43)基因的RAW264.7细胞系,探究GPR43基因在肺炎克雷伯菌感染过程中的作用机制。方法设计3对针对GPR43基因的小导向RNA(sgRNA),将sgRNA插入pLenticrisprV2质粒中,利用慢病毒包装系统包装含有sgRNA的重组质粒pLenticrisprV2;将病毒感染RAW264.7细胞,嘌呤霉素筛选单克隆细胞;将扩增的单克隆细胞提取基因组DNA,测序GPR43基因相关序列并与野生型GPR43基因进行比对,确认敲除成功的细胞株(GPR43^-/-RAW264.7细胞),Western blot法检测GPR43蛋白水平。肺炎克雷伯菌感染GPR43^-/-RAW264.7细胞,实时定量PCR法检测细菌感染后细胞内白细胞介素1β(IL-1β)、IL-6和肿瘤坏死因子α(TNF-α)表达水平的变化,并观察敲除GPR43后,RAW264.7细胞吞噬能力的变化。结果挑选的单克隆细胞经Western blot验证GPR43蛋白不表达,并且DNA测序结果显示在sgRNA插入位置缺失34个碱基,证明成功敲除GPR43基因。GPR43^-/-RAW264.7细胞感染肺炎克雷伯菌后,细胞中的IL-1β、IL-6和TNF-α表达水平均低于对照组,且GPR43^-/-RAW264.7细胞对肺炎克雷伯菌的吞噬能力降低。结论CRISPR/Cas9技术敲除GPR43基因,抑制其对肺炎克雷伯菌的吞噬功能及炎性细胞因子的产生。

Objective To construct cell line RAW264.7 with stable knockout of GPR43 gene using CRISPR/Cas9 system,and explore the role and mechanism of GPR43 gene in Klebsiella pneumoniae infection.Methods Three pairs of small-guide RNA(sgRNA)targeting the GPR43 gene were designed and inserted into plasmid pLenticrisprV2.The recombinant plasmid pLenticrisprV2 containing sgRNA was packaged using a lentivirus packaging system.RAW264.7 cells were transfected with viruses,and monoclonal cells were screened using puromycin.The genomic DNA was extracted from the amplified monoclonal cells.The GPR43 gene-related sequences were sequenced and compared with the wild-type GPR43 gene to confirm the cell line with successful knockout(GPR43^-/-RAW264.7 cells).The expression of GPR43 protein was detected by Western blotting.After GPR43^-/-RAW264.7 cells were transfected with Klebsiella pneumoniae,the changes in the expression of interleukin-1β(IL-1β),IL-6 and tumor necrosis factor-α(TNF-α)in the cells were detected using real-time quantitative PCR.Additionally,the phagocytic capacity of RAW264.7 cells after GPR43 knockout was observed.ResultsWestern blotting confirmed that GPR43 protein was not expressed in the selected monoclonal cells,and DNA sequencing showed that 34 bases were missing at the insertion site of sgRNA,which proved that GPR43 gene was successfully knocked out.After GPR43^-/-RAW264.7 cells were transfected with Klebsiella pneumoniae,the levels of IL-1β,IL-6 and TNF-αexpression in the cells were all lower than those in the control group,and the phagocytic capacity of GPR43^-/-RAW264.7 cells to Klebsiella pneumoniae decreased.Conclusion CRISPR/Cas9-based knockout of GPR43 gene in RAW264.7 cells can inhibit their phagocytosis for Klebsiella pneumoniae and production of inflammatory cytokines.