Description:
NFkBResponsiveLuciferaseReporterNIH/3T3StableCellLineisderivedfrommousefibroblast,andstablyexpressfireflyluciferasereportergeneunderthecontroloftheNFkB Responseelement. ThiscelllineisanidealcellularmodelformonitoringtheactivationofNFkBReceptorSignalingPathwaytriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.Principle:
NFkBplaysanimportantroleincontrollingmanyBIOLOGicalprocessesincludingimmuneandinflammatoryresponses,developmentalprocesses,cellulargrowth,andapoptosis.Inresponsetothevariousstimuli,suchasstress,cytokines,freerADIcals,ultravioletirradiation,andbacterialorviralantigens,NFkBisactivatedandtranslocatesfromcytoplasmtonucleus,whereNFkBbindstoitsresponseelementonthepromoterregionandregulatesawidespectrumofgeneexpression.DysfunctionofNFkBactivityisassociatedwithcancer,inflammatoryandautoimmunedisease,andviralinfection.MonitoringtheNFkBactivityisessentialtounveilthemechanismofthesediseasesandconductdrugdiscovery.SignosishasestablishedaNFkBluciferasereporterstablecelllinethathasbeenstablytransfectedwithpTA-NFkB-luciferasereportervectorand canbeusedasareportersystemformonitoringtheactivationofNFkBtriggeredbystimulitreatment suchasTNFα,IL-1αandIL-1β,enforcedgeneexpressionandgeneknockdown.
Thecelllinewasestablishedbytransfection usinga pTA-NFkB-luciferasereportervector,whichcontains4repeatsofNFkBbindingsites,aminimalpromoterupstreamofthefireflyluciferasecodingregion, alongwithhygromycinexpressionvectorfollowedbyhygromycinselection. ThehygromycinresistantclonesweresubsequentlyscreenedforTNFa-inducedluciferaseactivity.
PrinciplebehindTFluciferasereporter. TFluciferasereporterstablecelllineutilizesartificialpromoterconstructstodriveluciferaseexpression. Thepromoterregioncanconsistsofmultiplerepeatsofacis-elementTFbindingsite,aDNAfragmentfromthepromoterregionofaknownTFdownstreamgene,oraDNAfragmentcontainingputative/knownTFbindingsites. ThereareseveralwaysthataTFcanbeactivated,suchasthroughextracellularstimuliorthroughintracellularsignalingpathways. Onceactivated,theTFtranslocatestothenucleusandofteninteractswithrelevantco-factorstodrivegeneexpression. Onceluciferaseisexpressed,itcangeneratelightinanenzymaticassayandtheamountoflightmeasuredispositivelycorrelatedwiththelevelofTFactivation. |
Data:
AnalysisofSL-0006NFκBreporteractivityinresponsetovarioustreatments. Thecellswereseededona96-wellplateforovernightwithDMEMincluding10%FBS.ThecellsthenweretreatedwiththefollowingchemicalsinDMEMand0.1%FBSfor16hours:10ng/mLIL-1a,,10ng/mlPMA,10ng/mloncostatinM(OSM),100ng/mlIFN-gamma,and2ng/mlTNFα. NIH/3T3-NFkBLuciferaseReporterCellLineexhibitsresponsetoTNFαandIL-1awhencomparedtountreatedcells.
