Description:
NFkB ResponsiveLuciferaseReporterMEFCellLine isderivedfrommurineembryonicfibroblast,andstablyexpressfireflyluciferasereportergeneunderthecontrolofNFkBresponseelement. ThiscelllineisanidealcellularmodelformonitoringtheactivationofNFkBSignalingPathwaytriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.Principle:
NFkBplaysanimportantroleincontrollingmanyBIOLOGicalprocessesincludingimmuneandinflammatoryresponses,developmentalprocesses,cellulargrowth,andapoptosis.Inresponsetothevariousstimuli,suchasstress,cytokines,freerADIcals,ultravioletirradiation,andbacterialorviralantigens,NFkBisactivatedandtranslocatesfromcytoplasmtonucleus,whereNFkBbindstoitsresponseelementonthepromoterregionandregulatesawidespectrumofgeneexpression.DysfunctionofNFkBactivityisassociatedwithcancer,inflammatoryandautoimmunedisease,andviralinfection.MonitoringtheNFkBactivityisessentialtounveilthemechanismofthesediseasesandconductdrugdiscovery.
SignosishasestablishedaNFkBluciferasereporterstablecelllinethathasbeenstablytransfectedwithpTA-NFkB-luciferasereportervectorandcanbeusedforstudyingNFkBsignalingpathwaysactivatedbydifferentcytokines,suchasTNFαandmanyotherstimuli,enforcedgeneexpressionandgeneknockdown.ThecelllinewasestablishedbytransfectionusingapTA-NFkB-luciferasereportervector,whichcontains4repeatsofNFkBbindingsites,aminimalpromoterupstreamofthefireflyluciferasecodingregion,alongwithhygromycinexpressionvectorfollowedbyhygromycinselection.ThehygromycinresistantclonesweresubsequentlyscreenedforTNFa-inducedluciferaseactivity.
PrinciplebehindTFluciferasereporter. TFluciferasereporterstablecelllineutilizesartificialpromoterconstructstodriveluciferaseexpression. Thepromoterregioncanconsistsofmultiplerepeatsofacis-elementTFbindingsite,aDNAfragmentfromthepromoterregionofaknownTFdownstreamgene,oraDNAfragmentcontainingputative/knownTFbindingsites. ThereareseveralwaysthataTFcanbeactivated,suchasthroughextracellularstimuliorthroughintracellularsignalingpathways. Onceactivated,theTFtranslocatestothenucleusandofteninteractswithrelevantco-factorstodrivegeneexpression. Onceluciferaseisexpressed,itcangeneratelightinanenzymaticassayandtheamountoflightmeasuredispositivelycorrelatedwiththelevelofTFactivation. |
Data:
AnalysisofSL-0012NFκBreporteractivityinresponsetovarioustreatments. TheMEFcellswereseededona96-wellplateforovernightwithDMEMincluding10%FBS.ThecellsthenweretreatedwiththefollowingchemicalsinDMEMand0.1%FBSfor16hours:20ng/mlTNFα,10ng/mlPMA,100ng/mlIFN-gamma,10ng/mloncostatinM(OSM),and10ng/mLIL-1a. HEK293-NFkBLuciferaseReporterCellLineexhibitsresponsetoTNFαandIL-1awhencomparedtountreatedcells.
