Construction Of DF1 Cell Line Without Mda5 Function

As an important zoonosis,avain influenza has a great impact on breeding industry and public health all the time.Although H9N2 avian influenza virus does not causes high mortality after poultry infection,the immunity of infected chicken is reduced,and leads them to more susceptible to secondary infections.These cause a great economic loss of poultry industry.Upon avian Influenza virus(AIV)infection in mammals,viral RNA is recognized by RIG-I and MDA5.The activation of innate immunity leads to a cascade of downstream signaling pathways and results in the activation of IFN-I and a variety of inflammatory cytokines.It has been reported that RIG-I,which recognizes cytoplasmic viral RNA,is absent in chicken,while chickens are infected with avain influenza virus,they can also produce antiviral IFN-I in chickens.In the study of mammalian MDA5,it has been found that MDA5 can recognize viral RNA and induce IFN response in the process of anti-influenza virus infection.Although there is MDA5 receptor in chicken,its function in anti-influenza virus is not clear.In this study,we intended to construct mda5 gene inactivated cell line in DF1 cells via CRISPR/Cas9 technology,then evaluate the effect of H9N2 avian influenza virus induced IFN-β and the proliferation of the virus.In order to lay a foundation for the demonstration of avian influenza-induced IFN signaling pathway and the development of a suitable cell line for H9N2 influenza virus strains.In this study,we transfected the plasmid expressing Cas9 protein and transcribing sgRNA,but the results showed that none of the cells survived under the screening of antibiotics,that is,the method failed to constructed successfully.On the basis of optimizing the lentivirus construction system,the mutant cell line was constructed by lenti-CRISPR-v2 lentivirus system,but also failed.Therefore,we chose to use the double lentivirus system to construct the DF1 cell line,which expressed Cas9 protein stably through lentiCas9-blast lentivirus system.The results showed that the cell line was successfully constructed.Then,integrating the sgRNA into the cell line through lentivirus system of lentil-Guide-puro vector,and cell line function was verified by T7E1 assay and gene sequencing.The results showed that the constructed DF1-Cas9 cell line could play a function.In this study,we purified the mda5 inactivated cell line by IFN-sensitive VSV-GFP fluorescence virus,and the results showed that the cell line was successfully constructedCompared with IFN-β and PKR transcriptional levels were induced after H9N2 infection in DF1-?chmda5 and DF1-Cas9,the results showed that the DF1-Cas9 cell had an increased IFN-β transcription level post infection,as same as PKR,demonstrating that the innate immunity was activated in DF1-Cas9 cells.In addition,DF1 cells could still induce IFN-β in chmda5 knockout cells.However,the transcription level of IFN-β in chmda5 knockout cells was lower than that in DF1-Cas9 cells,indicating that chicken mda5 is not the only receptor for transcription of IFN-β and PKR in cells,there may be other signaling molecules involved in the transcriptional regulation of IFN-and PKR.Determination of hemagglutination titer of supernatant virus,the results showed that inactivation of mda5 could rapidly increase the proliferation of H9N2 virus within 24 hours.We have constructed the DF1 cell line expressing Cas9 protein by CRISPRR / Cas9 technique,and constructed a functionally inactive DF1 cell line based on DF1-Cas9 cell line.Inactivation of mda5 function in DF1 cells significantly down-regulates H9N2-induced transcription of IFN-β and increases H9N2 virus proliferation in a short period of time.