Description:
NFAT ResponsiveLuciferaseReporterJurkatT StableCellLineisderivedfromhumanTlymphocyte,andstablyexpressfireflyluciferasereportergeneunderthecontrolofNFATresponseelement. ThiscelllineisanidealcellularmodelformonitoringtheactivationofCalciumSignalingPathwaytriggeredbystimulitreatment,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:
AnalysisofNFATLuciferaseReporterJurkatTStableCellLine.Thecellswereseededon a96-wellplateinmediacontaining10ng/mlPMA,1uMionomycin,and0.1%FBSfor16hours. Morethan200foldincreaseinluciferaseactivitywasdetectedwhencomparedtountreatedcells.
