Description:
STAT1ResponsiveLuciferaseReporterHelaStableCellLineisderivedfromhumancervicalcancer,andstablyexpressfireflyluciferasereportergeneunderthecontroloftheSTAT1responseelement. ThiscelllineisanidealcellularmodelformonitoringtheactivationofJAK-STATReceptorSignalingPathwaytriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.Principle:
STAT1exertsacomplexarrayoffunctionsonbothtumorcellsandtheimmunesystemandisusuallyconsideredasatumorsuppressor.STAT1isacentralmediatoroftypeII(gamma)IFNs,afamilyofmultifunctionalsecretedproteinsinvolvedincellgrowthregulationandantiviralandimmunedefense.IFN-gamma(IFNγ),throughJAK1andJAK2,mainlytriggersprolongedSTAT1activationthatinducesgeneexpressionbybindingtogamma-activatedsequences(GAS). ThiscelllinecanbeusedtomonitortheactivationofStat1inresponsetothedifferentstimuli,suchasIFNgamma.
Thecelllinewasestablishedbytransfection usinga pTA-Stat1-luciferasereportervector,whichcontains4repeatsofStat1bindingsites,aminimalpromoterupstreamofthefireflyluciferasecodingregion, alongwithhygromycinexpressionvectorfollowedbyhygromycinselection. Thehygromycinresistantclonesweresubsequentlyscreenedforoncostatininducedluciferaseactivity.Theclonewiththehighestfoldinduction(50fold)wasselectedandexpandedtoproducethisstablecellline.
PrinciplebehindTFluciferasereporter. TFluciferasereporterstablecelllineutilizesartificialpromoterconstructstodriveluciferaseexpression. Thepromoterregioncanconsistsofmultiplerepeatsofacis-elementTFbindingsite,aDNAfragmentfromthepromoterregionofaknownTFdownstreamgene,oraDNAfragmentcontainingputative/knownTFbindingsites. ThereareseveralwaysthataTFcanbeactivated,suchasthroughextracellularstimuliorthroughintracellularsignalingpathways. Onceactivated,theTFtranslocatestothenucleusandofteninteractswithrelevantco-factorstodrivegeneexpression. Onceluciferaseisexpressed,itcangeneratelightinanenzymaticassayandtheamountoflightmeasuredispositivelycorrelatedwiththelevelofTFactivation. |
Data:
AnalysisofHeLa/STAT1-luciferaseactivity. Thecellswereseededona96-wellplateforovernightandthencellsweretreatedwithorwithout30ng/mlinterferon-gammainDMEMand0.1%FBSfor16hours. Interferon-gammainducesmorethan25foldincreaseinluciferaseactivitywhencomparedtountreatedcells.
