Description:
IRFResponsiveLuciferaseReporterHEK293CellLineisderivedfromhumanembryonickidney,andstablyexpressfireflyluciferasereportergeneunderthecontrolofIRFresponseelement.ThiscelllineisanidealcellularmodelformonitoringtheactivationofImmuneResponsePathway triggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.Principle:
Membersoftheinterferonregulatorytranscriptionfactor(IRF)familyareinvolvedinantiviraldefense,cellgrowthregulation,andimmuneactivation. InitiallyidentifiedasregulatorsoftypeIinterferon(IFN)geneinduction,IRFfamilyoftranscriptionfactorstransducesignalsformultipleclassesofthepattern-recognitionreceptors(PRRs),suchasToll-likereceptors,retinoicacid–inducIBLegene-I(RIG-I)-likereceptors(RLRs),NOD-likereceptors(NLRs),C-typelectinreceptors(CLRs),andothernucleicacid–sensingreceptors. Whenactivated,eachIRFproteintranslocatestothenucleusandbindstoDNAsequencesimilartoIFN-stimulatedresponseelement(ISRE). IRFactivationcanenhancetheIFN-mediatedantiviralimmuneresponse. Oppositely,abnormalactivationoftypeIIFNscontributestothedevelopmentofautoimmunediseases,suchasSLE. SignosishasestablishedanIRFluciferasereporterstablecelllinethatcanbeusedasareportersystemformonitoringtheactivationofIRFtriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.
Thecelllineisestablishedbytransfectionusinga pTA-GAS/ISRE-luciferasereportervector,whichcontainsIRFbindingsites,aminimalpromoterupstreamofthefireflyluciferasecodingregion, alongwithhygromycinexpressionvectorfollowedbyhygromycinselection. ThehygromycinresistantclonesweresubsequentlyscreenedforIFNgamma-inducedluciferaseactivity.
PrinciplebehindTFluciferasereporter. TFluciferasereporterstablecelllineutilizesartificialpromoterconstructstodriveluciferaseexpression. Thepromoterregioncanconsistsofmultiplerepeatsofacis-elementTFbindingsite,aDNAfragmentfromthepromoterregionofaknownTFdownstreamgene,oraDNAfragmentcontainingputative/knownTFbindingsites. ThereareseveralwaysthataTFcanbeactivated,suchasthroughextracellularstimuliorthroughintracellularsignalingpathways. Onceactivated,theTFtranslocatestothenucleusandofteninteractswithrelevantco-factorstodrivegeneexpression. Onceluciferaseisexpressed,itcangeneratelightinanenzymaticassayandtheamountoflightmeasuredispositivelycorrelatedwiththelevelofTFactivation. |
Data:
AnalysisofIRFLuciferaseReporterHEK293StableCellLine. TheHEK293cellswereseededona96-wellplateforovernightwithDMEMincluding10%FBS.Thecellsthenwereserum-deprivedfor8hoursbeforetreatingwiththefollowingchemicalsinDMEMand0.1%FBSfor16hours:100ng/mlIFN-gamma,20ng/mlTNFα,20ng/mLIL-1a,5ng/mLpolyI:C,10ng/mlPMA,10ng/mLTGFb,and50uMTBHQ. IRFLuciferaseReporterHEK293CellLineexhibitsmorethan20-foldresponsetoIFNgammawhencomparedtountreatedcells.
