The Establishment And Verification Of STING Gene Knockout Porcine Lung Macrophage Cell Line

The natural immune system is the first line of defense against the invasion of foreign microorganisms.STING?Stimulator of Interferon Genes?is an important linker protein in the natural immune signaling pathway.The intracellular second messenger cyclic guanosine monophosphate?adenosine monophosphate,c GAMP?is an endogenous activator of STING.The combination of both could activatethe STING/TBK1/IRF3 signaling pathway and ultimately activate the expression of type I interferon.In order to establish a screening tool for antiviral small molecule compounds based on STING as a target,this study established a STING gene knockout porcine lung macrophage cell line by using a lentiviral-mediated CRISPR/Cas9 genome editing technique.The recombinant plasmid pSTING-sgRNA constructed by designing single-stranded guide RNA?Single guide RNA,sg RNA?,was transfected into human embryonic kidney cells?Human embryonic kidney 293T/17,HEK293/T17?to reciceve lentivirus.The lentivirus collected from HEK293/T17 cells was used to infect porcine alveolar macrophages?3D4/21?and screened by Puromycin to obtain the STING gene knock-out polyclonal cell line.The STING gene knockout monoclonal(3D4/21-STING^-/-)cell line was obtained by limiting dilution method and sequenced.For the purpose of verifying whether 3D4/21-STING^-/-was established successfully,3D4/21-WT and 3D4/21-STING^-/-cells were first treated with different concentrations of STING agonist?G10?and infected with PRV-GFP,after 36 hours the difference in fluorescence intensity between the two cells was detected by fluorescence microscopy and flow cytometry;secondly,treatment of 3D4/21-WT and3D4/21-STING^-/-cells with G10 and infection with PRV-GFP and PRV-QXX was performed to examine the effect of STING gene knockout on the change of virus titer.Finally,3D4/21-WT and 3D4/21-STING^-/-were treated with G10,and the difference in m RNA expression level of Interferon stimulated gene?ISG?in two cell lines wasdetected by real-time fluorescence quantitative PCR.The test results showed that the pSTING-sgRNA recombinant plasmid was successfully constructed.After lentiviral vector-infected with the 3D4/21 cells were digested by T7 gene detected confirmed to have a significant effect on STING gene editing.It was further monoclonalized and identified by sequencing in order to obtain the 3D4/21-STING^-/-monoclonal cell line.First,3D4/21-WT and3D4/21-STING^-/-cells were treated with different concentrations of G10 and infected with PRV-GFP and PRV-QXX for fluorescence imaging,flow cytometry and virus titer determination.It was found that with the increase of G10 drug concentration,the fluorescence intensity of 3D4/21-WT cells showed a decreasing trend,and the fluorescence intensity of 3D4/21-STING^-/-cells did not change significantly;the titer of virus harvested by 3D4/21-WT cells was reduced,and there was no significant change in virus titer harvested by 3D4/21-STING^-/-cells.Finally,the real-time fluorescence quantitative PCR was used to detect 3D4/21-WT and 3D4/21-STING^-/-cells with G10 pretreatment.The results showed that the expression levels of ISG15 and ISG20 were significantly increased in 3D4/21-WT cells as the concentration of G10 was increased.However,there was no significant change in m RNA expression of ISG15 and ISG20 in 3D4/21-STING^-/-cells.The above results indicated that the3D4/21 STING knockout monoclonal cell line was successfully constructed.This study would provide techniques for the screening of anti-viral small molecule compounds based on the STING signaling pathway and provide new strategies for the prevention and treatment of pseudorabies.