Description:
CREBResponsiveLuciferaseReporterHEK293StableCellLineisderivedfromhumanembryonickidney,and stablyexpressfireflyluciferasereportergeneunderthecontrolofCREBresponseelement.ThiscelllineisanidealcellularmodelformonitoringtheactivationofcAMP,PKA,CaMKSignaling ReceptorSignalingPathwaytriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.Principle:
CyclicAMPresponseelement(CRE)-bindingprotein(CREB)isatranscriptionfactorthatregulatesandrespondstodiversecellularresponses,rangingfromproliferation,survival,differentiation,stressresponses,andneuronalactivity. Thesecellularsignalsleadtoupstreamkinaseactivation,suchasproteinkinaseA(PKA),pp90ribosomalS6kinase(pp90RSK),andCa2+/calmodulin-dependentproteinkinases(CaMKs),andthesekinasesinturnphosphorylateCREBtoinduceCREBactivity. CREBincreasesthetranscriptionofgenesthatcontaincAMPresponsiveelements.
SignosishasdevelopedCREBluciferasereporterstablecelllinebyco-transfectingCREBluciferasereportervectorandhygromycinexpressionvector. Thehygromycinresistantclonesweresubsequentlyscreenedforforskolin-inducedluciferaseactivity. ThecelllinecanbeusedasareportersystemformonitoringtheactivationofCREBtriggeredbystimulitreatment,suchasforskolinandgeneoverexpressionandgeneknockdown.
PrinciplebehindTFluciferasereporter. TFluciferasereporterstablecelllineutilizesartificialpromoterconstructstodriveluciferaseexpression. Thepromoterregioncanconsistsofmultiplerepeatsofacis-elementTFbindingsite,aDNAfragmentfromthepromoterregionofaknownTFdownstreamgene,oraDNAfragmentcontainingputative/knownTFbindingsites. ThereareseveralwaysthataTFcanbeactivated,suchasthroughextracellularstimuliorthroughintracellularsignalingpathways. Onceactivated,theTFtranslocatestothenucleusandofteninteractswithrelevantco-factorstodrivegeneexpression. Onceluciferaseisexpressed,itcangeneratelightinanenzymaticassayandtheamountoflightmeasuredispositivelycorrelatedwiththelevelofTFactivation. |
Data:
AnalysisofSL-0020CREBreporteractivityinresponsetoforskolintreatment. Thecellswereseededona96-wellplateforovernightwithDMEMincluding10%FBS.Thecellsthenweretreatedwithorwithout10uMforkoslininDMEMand0.1%FBSfor16hours. Forskolininducedover50-foldincreaseinluciferaseactivitycomparedtountreatedcells.
