Description:
NFATResponsiveLuciferaseReporterNIH/3T3StableCellLineisderivedfrommousefibroblast,andstablyexpressfireflyluciferasereportergeneunderthecontrolofNFATresponseelement. Thiscelllineisanidealcellularmodelformonitoringtheactivation ofCalciumSignalingPathwaytriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.Principle:
NFATsareafamilyoftranscriptionalfactorsthatplayanimportantroleinimmuneresponseaswellasinthedevelopmentofcardiac,skeletalmuscle,andnervoussystems. NFATsareregulatedbycalciumsignaling.Throughcalciumactivationofthephosphatasecalcineurin,NFATcproteinstranslocatefromthecytoplasmintothenucleus,wheretheycooperatewithotherproteinstomediategeneexpression.NuclearimportofNFATisblockedbykinasessuchasPKAandGSK3.NFATsarealsoimplicatedinbreastcancer. SignosishasestablishedaNFATluciferasereportercelllinethathasbeenstablytransfectedwithaNFAT-luciferasereporterconstruct.Viatheanalysisofluciferase,thecelllinecanbeemployedtomonitorthecellularchangesofNFATactivitiesthataretriggeredbystimulation,compoundtreatment,enforcedgeneexpressionorgeneknockdown.
Thecelllineisestablishedbytransfectionusinga pTA-NFAT-luciferasereportervector,whichcontainsNFATbindingsites,aminimalpromoterupstreamofthefireflyluciferasecodingregion, alongwithhygromycinexpressionvectorfollowedbyhygromycinselection. ThehygromycinresistantclonesweresubsequentlyscreenedforPMA+ionomycin-inducedluciferaseactivity.
PrinciplebehindTFluciferasereporter. TFluciferasereporterstablecelllineutilizesartificialpromoterconstructstodriveluciferaseexpression. Thepromoterregioncanconsistsofmultiplerepeatsofacis-elementTFbindingsite,aDNAfragmentfromthepromoterregionofaknownTFdownstreamgene,oraDNAfragmentcontainingputative/knownTFbindingsites. ThereareseveralwaysthataTFcanbeactivated,suchasthroughextracellularstimuliorthroughintracellularsignalingpathways. Onceactivated,theTFtranslocatestothenucleusandofteninteractswithrelevantco-factorstodrivegeneexpression. Onceluciferaseisexpressed,itcangeneratelightinanenzymaticassayandtheamountoflightmeasuredispositivelycorrelatedwiththelevelofTFactivation. |
Data:
AnalysisofNFATLuciferaseReporterNIH/3T3StableCellLine. Thecellswereseededona96-wellplateforovernightwithDMEMincluding10%FBS.Thecellsthenweretreatedwithorwithout10ng/mlPMAand1uMionomycininDMEMand0.1%FBSfor16hours. Morethan5foldincreaseinluciferaseactivitywasdetectedwhencomparedtountreatedcells.
