Description:
NFkB ResponsiveLuciferaseReporterHelaStableCellLineisderivedfromhumancervicalcancer,andstablyexpressfireflyluciferasereportergeneunderthecontroloftheNFkBresponseelement.Thiscelllineisanidealcellularmodelformonitoringtheactivation ofNFkBPathwaytriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.Principle:
NFkBplaysanimportantroleincontrollingmanyBIOLOGicalprocessesincludingimmuneandinflammatoryresponses,developmentalprocesses,cellulargrowth,andapoptosis.Inresponsetothevariousstimuli,suchasstress,cytokines,freerADIcals,ultravioletirradiation,andbacterialorviralantigens,NFkBisactivatedandtranslocatesfromcytoplasmtonucleus,whereNFkBbindstoitsresponseelementonthepromoterregionandregulatesawidespectrumofgeneexpression.DysfunctionofNFkBactivityisassociatedwithcancer,inflammatoryandautoimmunedisease,andviralinfection.MonitoringtheNFkBactivityisessentialtounveilthemechanismofthesediseasesandconductdrugdiscovery.SignosishasestablishedanNFkBluciferasereporterstablecelllinethathasbeenstablytransfectedwithpTA-NFkB-luciferasereportervector,whichcontains4repeatsofNFkBbindingsites,aminimalpromoterupstreamofthefireflyluciferasecodingregion. Therefore,thecelllinecanbeusedasareportersystemformonitoringtheactivationofNFkBtriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown. ThestablecelllinecanbeusedforstudyingNFkBsignalingpathwaysactivatedbydifferentcytokines,suchasTNFa, andmanyotherstimuli.
Thecelllinewasestablishedbytransfectionusinga pTA-NFkB-luciferasereportervector,whichcontains4repeatsofNFkBbindingsites,aminimalpromoterupstreamofthefireflyluciferasecodingregion, alongwithhygromycinexpressionvectorfollowedbyhygromycinselection.ThehygromycinresistantclonesweresubsequentlyscreenedforTNFa-induced(orIL-1)luciferaseactivity.
PrinciplebehindTFluciferasereporter. TFluciferasereporterstablecelllineutilizesartificialpromoterconstructstodriveluciferaseexpression. Thepromoterregioncanconsistsofmultiplerepeatsofacis-elementTFbindingsite,aDNAfragmentfromthepromoterregionofaknownTFdownstreamgene,oraDNAfragmentcontainingputative/knownTFbindingsites. ThereareseveralwaysthataTFcanbeactivated,suchasthroughextracellularstimuliorthroughintracellularsignalingpathways. Onceactivated,theTFtranslocatestothenucleusandofteninteractswithrelevantco-factorstodrivegeneexpression. Onceluciferaseisexpressed,itcangeneratelightinanenzymaticassayandtheamountoflightmeasuredispositivelycorrelatedwiththelevelofTFactivation. |
Data:
AnalysisofTNFa/NFkBPathwayReporterCellLineinresponsetoTNFatreatment.
Thecellswereseededon a96-wellplateforovernightwithDMEMincluding10%FBS.Thecellthenweretreatedwith20ng/mlTNFainDMEM+0.1%FBSfor6hours.
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