Description:
GRResponsiveLuciferaseReporterHelaStableCellLineisderivedfromhumancervicalcancer,andstablyexpressfireflyluciferasereportergeneunderthecontrolofGRresponseelement.Thiscelllineisanidealcellularmodelformonitoringtheactivation ofGlucocorticoidSignalingReceptorSignalingPathwaytriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.Principle:
TheGlucocorticoidreceptor(GR)isamajorplayerindevelopment,metabolismandimmuneresponse. Whenactivatedbystimuli,GRtranslocatesfromthecytoplasmintothenucleus,andthenbindsaDNArecognitionsitetoregulategeneexpression. Inaddition,activatedGRcantransrepressothertranscriptionfactorsthathavebeenmisregulatedincancerandotherdiseases.
SignosishasestablishedaGRluciferasereporterstablecellline,bytransfectionofaGRfireflyluciferasereportervectoralongwitheitherG418orhygromycinexpressionvectorfollowedbyG418orhygromycinselection,respectively.Theantibioticresistantclonesweresubsequentlyscreenedfordexamethasone(DEX)-inducedluciferaseactivity.Theclonewiththehighestfoldinductionwasselectedandexpandedforproduction.
PrinciplebehindTFluciferasereporter. TFluciferasereporterstablecelllineutilizesartificialpromoterconstructstodriveluciferaseexpression. Thepromoterregioncanconsistsofmultiplerepeatsofacis-elementTFbindingsite,aDNAfragmentfromthepromoterregionofaknownTFdownstreamgene,oraDNAfragmentcontainingputative/knownTFbindingsites. ThereareseveralwaysthataTFcanbeactivated,suchasthroughextracellularstimuliorthroughintracellularsignalingpathways. Onceactivated,theTFtranslocatestothenucleusandofteninteractswithrelevantco-factorstodrivegeneexpression. Onceluciferaseisexpressed,itcangeneratelightinanenzymaticassayandtheamountoflightmeasuredispositivelycorrelatedwiththelevelofTFactivation. |
Data:
AnalysisofGRLuciferaseReporterHelacellline. Thecellswereseededona96-wellplateforovernightwithDMEMincluding10%FBS.Thecellsthenweretreatedwithorwithout2uMDexamethasone(DEX)respectivelyinDMEMand0.1%FBSfor16hours. Closeto100foldincreaseinluciferaseactivitywasdetectedwhencomparedtountreatedcells.
