Description:
p53ResponsiveLuciferaseReporterHelaStableCellLineisderivedfromhumancervicalcancer ,andstablyexpressfireflyluciferasereportergeneunderthecontrolofthep53responseelement. Thiscelllineisanidealcellularmodelformonitoringtheactivationofp53ReceptorSignalingPathwaytriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.Principle:
Thep53pathwayplaysacrucialroleineffectivetumorsuppressionbecauseofitscentralfunctionincellcycleregulation,DNArepair,cellularsenescence,andapoptosis,whichcanbeusedforpotentiallydevelopnewdrugtherapiesagainstcancer.UponactivationbyDNAdamage,oncogeneactivation,orhypoxia,p53bindstoitsDNArecognitionsiteonthepromoterregionsofthetargetgenesandregulatethegeneexpression. Signosishasdevelopedp53luciferasereporterHelastablecellline,inwhichluciferaseactivityisspecificallyassociatedwiththeactivityofp53.Therefore,thecelllinecanbeusedasareportersystemformonitoringtheactivationofp53triggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.
Thecelllinewasestablishedbytransfection usinga pTA-p53-luciferasereportervector,whichcontains6repeatsofp53bindingsites,aminimalpromoterupstreamofthefireflyluciferasecodingregion, alongwithhygromycinexpressionvectorfollowedbyhygromycinselection. Thehygromycinresistantclonesweresubsequentlyscreenedfordoxorubicin-inducedluciferaseactivity.
PrinciplebehindTFluciferasereporter. TFluciferasereporterstablecelllineutilizesartificialpromoterconstructstodriveluciferaseexpression. Thepromoterregioncanconsistsofmultiplerepeatsofacis-elementTFbindingsite,aDNAfragmentfromthepromoterregionofaknownTFdownstreamgene,oraDNAfragmentcontainingputative/knownTFbindingsites. ThereareseveralwaysthataTFcanbeactivated,suchasthroughextracellularstimuliorthroughintracellularsignalingpathways. Onceactivated,theTFtranslocatestothenucleusandofteninteractswithrelevantco-factorstodrivegeneexpression. Onceluciferaseisexpressed,itcangeneratelightinanenzymaticassayandtheamountoflightmeasuredispositivelycorrelatedwiththelevelofTFactivation. |
Data:
Analysisofp53ReporterHelaStableCellLineinresponsetostimuli. Thecellswereseededona96-wellplateforovernightwithDMEMincluding10%FBS.Thecellsthenwereeithertreatedwith500ng/mlDoxorubicinor2ug/mlQuinacrinerespectivelyinDMEMand10%FBSfor16hours.
