Description:
NFAT ResponsiveLuciferaseReporterJurkatTStableCellLineisderivedfromhumanTlymphocyte,andstablyexpressfireflyluciferasereportergeneunderthecontrolofNFATresponseelement. ThiscelllineisanidealcellularmodelformonitoringtheactivationofCalciumSignalingPathwaytriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.Principle:
TcellreceptorsignalingplaysanimportantroleinTcellactivation,proliferationanddifferentiationleADIngtotheinitiationseriestyrosinephosphorylationeventsthattriggermultiplesignalingpathways,oneofwhichistheactivationofCa2+/proteinkinaseCpathways.TheincreasedintracellularcalciumlevelsandtheactivationofPKCsleadstotheactivationthenuclearfactorofactivatorTcells(NFAT)transcriptionfactorresultingindownstreamgeneexpression.JurkatleukaemicT-celllineiswidelyused celllinefor studyingTCR/NFATsignaling. Signosis’JurkatNFATReporterStableCellLineisestablishedbytransfectionusinga pTA-NFAT-luciferasereportervector,whichcontainsNFATbindingsites,aminimalpromoterupstreamofthefireflyluciferasecodingregion, alongwithhygromycinexpressionvectorfollowedbyhygromycinselection. ThehygromycinresistantclonesweresubsequentlyscreenedforluciferaseactivityinducedbyPMA+ionomycin.
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.
