The Establishment Of IRF3 Gene Knockout Porcine Epithelial Cell Line

Innate immunity is the natural immune system that lays the foundation for immune response and regulation for the differentiation and identification of self-and non-self components.Interferon regulatory factor 3(IRF3)is one of the main members of interferon regulatory factor family.It is also an important interferon regulator in innate immune signaling pathway.When cells is infected by DNA virus,the STING/TBKI/IRF3 innate immune signaling pathway and the expression of type ? interferon(IFN)are finally activated.In this study,lentivirus-mediated CRISPR/Cas9 genome editing technique was used to construct IRF3 stably knockout porcine kidney epithelial PK15 cell line.pIRF3-sgRNA recombinant plasmid was constructed and co-transfected with lentivirus packaging vector into HEK293T/17 cells.Lentivirus was harvested and infected with PK15 cells.The polyclonal cell line was obtained by purinomycin screening.T7 restriction endonuclease digestion was used to identify polyclonal cells with high genome editing efficiency.Then PK15-IRF3-/-monoclonal stable cell line was obtained by limited dilution method.In order to verify whether the stable knockout cell line of IRF3 gene was successfully constructed,Firstly,fluorescence microscopy and flow cytometry were used to observe PRV-GFP replication in PK15-WT and PK15-IRF3-/-cells at 0,6,12,24,36 and 48 hours porst infection.Secondly,the titer of PRV-QXX in PK15-WT and PK15-IRF3-/-cells was detected by TCID50 assay at 0,6,12,24,36 and 48 hours porst infection.The expression of PRV-QXX gE protein in PK15-WT and PK15-IRF3-/-cells was detected by western blot at 0,6,12,24,36 and 48 hours porst infection.Finally,the expression of PRV-QXX TK and IFN-? mRNA in PK15-WT and PK15-IRF3-/-cells were detected by real-time fluorescence quantitative PCR at 0,6,12,24,36 and 48 hours porst infection.The results showed that the recombinant plasmid of IRF3-sgRNA was successfully constructed.PK15 cells infected with the lentivirus showed a significant editing effect on the IRF3 gene analyzed by T7 restriction endonuclease digestion.The monoclonal cells were further obtained and identified by DNA sequencing.Fluorescence microscopy and flow cytometry results showed that the fluorescence intensity in PK15-IRF3-/-cells was increased with time and was significantly stronger than that in PK15-WT cells infected with PRV-GFP.After infection with PRV-QXX,virus titer in PK15-IRF3-/-was significantly increased with time compared with that in PK15-WT cells,and the expression level of PRV gE protein was also significantly increased with time than that in PK15-WT cells.The expression of PRV-QXX TK mRNA in PK15-IRF3-/-cells was significantly higher than that in PK15-WT cells.The expression of IFN-? mRNA in PK15-WT cells was significantly increased with time,but there was no significant change in PK15-IRF3-/-cells.The above results indicate that IRF3 plays an important role in PRV replication,and knockout of IRF3 significantly promotes the proliferation of PRV.These results indicate that the IRF3 knockout pig kidney epithelial cell line is successfully constructed.This cell line provides an effective tool for further study of the function of IRF3.