The Effects Of CGAS-STING-Mediated Immune Pathway Inhibitors On The Transfection Efficiency

DNA transfection is one of the major methods used for molecular biology research and for gene therapy.However,the DNA transfection efficiency in different cell types is different and the transfection efficiency in many primary cells is especially low.Therefore,it is important for both molecular biology research and gene therapy to improve the DNA transfection eifficiency.In DNA transfection,after the exogenous DNA enters the cell,it may be distributed to the cytoplasm,thus causes immune activation of the cGAS-STING mediated DNA-sensing pathway.At present,most studies are focused on improving transfection efficiency by optimizing transfection conditions and developing transfection media,while few have linked the transfection efficiency with the cGAS-STING mediated DNA immune recognition pathway.Therefore,this thesis studied the influence of cGAS-STING mediated DNA immune recognition pathway on DNA transfection process,and explored ways to improve the transfection efficiency.We found that plasmid DNA transfection can activate cGAS-STING mediated DNA immune responses,while cGAS plays an important role in it since cGAS deletion can improve cell transfection efficiency.Then we constructed the type I interferon receptor(IFNAR1)knockout cell line,and verified that the suppression of interferon(IFN)mediated downstream immune response could affect the immune response induced by DNA transfection.Based on these studies,we have used cGAS-STING downstream TBK1 inhibitors and the inhibitors targeting type I IFN-mediated downstream pathway factors JAK1/2 and JAK35 on several different cells for further experiments.The results showed that the effects of these inhibitors on transfection efficiency were similar to the results of cGAS and IFNAR1 knockout strains,and were not affected by the transfection methods.These results suggest that cGAS-STING-mediated IFN immune response pathway plays an important role in the regulation of DNA transfection.By inhibiting IFN-mediated immune response,DNA transfection efficiency can be increased.These results provide a solid foundation for further investigation on the mechanism of DNA transfection and the development of efficient DNA transfection methods.