Using The CRISPR/Cas9 Technology To Build The Interferon Receptor Knockout Cell Line And Function Study

Since Interferon(IFN)was discovered and proved in the cultured cells that can inhibit the virus infection has passed more than half a century.Since then,researchers have been able to elucidate the molecular mechanisms of IFN signaling pathways,gradually the pleiotropic effects of classification of them in the cell,use them for a variety of diseases with potential therapeutic effect.Although progress is abundant,but there are some basic problems to be solved,there are a lot of complexity can't figure out.IFNs is a kind of cytokines and be divided into three types of:?,? and ?type.They have different expression patterns,has many roles in innate and acquired immunity.The current study,the ? and ? antiviral property is the most studied.The ?and ? interferon signaling pathways has interesting features which is most proteins in the same receptor complex deliver signal.In the human body,type ? interferons,including 13 interferons a gene,and single gene IFN-?,IFN-?,IFN-?,IFN-?.All through a total of 17 ? type interferon contains a single IFNAR1 and IFNAR2 dimers receptor complexes and combine with them and signal,they are almost exists in all of the nucleated cells.Compared with most type ? IFNs,there is only one type ?interferons,IFN-?,mainly produced by immune cells.IFN-? through a four polymers is composed of two subunits IFNGR1 and IFNGR2 composites of homologous dimers.Similar to IFNs ? type,type ?FN receptor is widely distributed in the tissue,as a result,almost all of the cells are capable of response type ? interferons.Type ?IFNs(also called interferon X)including IFNL1,IFNL2,IFNL3 and recently newly discovered IFNL4.Type ? IFNs signal by including single IL-10R2 and IFNLR1 dimers receptor complexes.Gene editing techniques have been used to study gene function becomes a powerful tool for people,while the CRISPR/Cas9 is following the ZFN and TALEN development in recent years,which is the rapid development of gene editing techniques.ZFN is by restriction endonucleases Fok I shear DNA components and with multiple zinc finger protein arrays of DNA binding element hybrid,two Fok I monomer is closely near to dimer formation,play a catalytic activity and the formation of double chain fracture;TALEN is a pair of can be in the series of specific recognition of DNA fragments,amino acid repeat sequences guide endonuclease Fok I,it works on targeting double-stranded DNA,the polymerization of Fok I can show enzyme function,causing double chain fracture;CRISPR/Cas9 is identified by a specific targeted point guided gRNA Cas9 endonuclease,cutting target points form a double chain rupture.Double chain fracture cause DNA repair,repair mechanisms have NHEJ(Non-homologous End Joining)or HR(Homologous Recombination),after repair,will produce the product,such as lack of mutation,insert,insert genes that cause,such as gene knockout results.This gene editing technique has wide applicable range,high efficiency,low cost,and the advantages of short cycle,has been in the mouse,zebrafish,human genes,bacteria,viruses,plants,and many other successful application in the species,and show a strong clinical treatment and great potential in the field of human and animal husbandry.Using the CRISPR/Cas9 technology in human cancer cells for gene knock out,mainly includes the following steps:acquire purpose targeted gene,choose appropriate targeted gene sequences point and design the best primer,build gRNA in vitro transcription carrier,lentivirus packaging,screening with labels and pick monoclonal cell and functional verification.This research use the CRISPR/Cas9 technology in Huh7 liver cancer cell and A549 lung cancer cell and respectively knockout IFNAR1 and IL28R1 the two interferon receptor,have been obtained respectively IFNAR1-KO and IL28R1-KO knockout cell lines,and is verified at the protein level,clean completely,background without antibody detection.Again to knock out cell line as the carrier,in which the interleukin 27 and interleukin 32 antiviral research,based on the conclusion from in RNA levels were measured in three RNA replication of IAV and the secretion of HBV virus E and S antigen in both IFNAR1 or IL28R1 cell receptor knockout,IL-27 and IL-32 itself antiviral effect disappeared,shows that both of them play a role on antiviral by interferon pathway and two types depend on.Taken together,our findings is lack of interferon receptor on cell response to weaken against virus invasion,human's first line of defense is attacked,and may lead to a series of signal response in cells in response to this invasion,for this,give us the back of the research provides a train of thought.