Description:
NFkBResponsiveLuciferaseReporterHepG2StableCellLineisderivedfromhumanlivercancer,andstablyexpressfireflyluciferasereportergeneunderthecontroloftheNFkB responseelement. Thiscelllineisanidealcellularmodelformonitoringtheactivation ofNFkBReceptorSignalingPathwaytriggeredbystimulitreatment,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-0017NFκBreporteractivityinresponsetoTNFαtreatment. TheHepG2cellswereseededona96-wellplateforovernightwithDMEMincluding10%FBS.Thecellsthenweretreatedwithorwithout20ng/mlTNFαinDMEMand0.1%FBSfor16hours. HepG2-NFkBLuciferaseReporterCellLineexhibitsTNFα-dependentincreaseinluciferaseactivitywhencomparedtountreatedcells.
