Description:
p53 ResponsiveLuciferaseReporterRKOStableCellLineisderivedfromhumancoloncancer,andstablyexpressfireflyluciferasereportergeneunderthecontrolofthep53responseelement.Thiscelllineisanidealcellularmodelformonitoringtheactivationofp53Pathwaytriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.Principle:
Thep53pathwayplaysacrucialroleineffectivetumorsuppressionbecauseofitscentralfunctionincellcycleregulation,DNArepair,cellularsenescence,andapoptosis,whichcanbeusedforpotentiallydevelopnewdrugtherapiesagainstcancer.UponactivationbyDNAdamage,oncogeneactivation,orhypoxia,p53bindstoitsDNArecognitionsiteonthepromoterregionsofthetargetgenesandregulatethegeneexpression. Signosishasestablishedp53luciferasereporterstablecellline,inwhichluciferaseactivityisspecificallyassociatedwiththeactivityofp53.Therefore,thecelllinecanbeusedasareportersystemformonitoringtheactivationofp53triggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.
Thecelllinewasestablishedbytransfectionofp53luciferasereportervectoralongwithG418expressionvectorfollowedbyG418selection.TheG418resistantclonesweresubsequentlyscreenedforetoposide-inducedluciferaseactivity.Theclonewiththehighestfoldinduction(35fold)wasselectedandexpandedtoproducethisstablecellline.
PrinciplebehindTFluciferasereporter. TFluciferasereporterstablecelllineutilizesartificialpromoterconstructstodriveluciferaseexpression. Thepromoterregioncanconsistsofmultiplerepeatsofacis-elementTFbindingsite,aDNAfragmentfromthepromoterregionofaknownTFdownstreamgene,oraDNAfragmentcontainingputative/knownTFbindingsites. ThereareseveralwaysthataTFcanbeactivated,suchasthroughextracellularstimuliorthroughintracellularsignalingpathways. Onceactivated,theTFtranslocatestothenucleusandofteninteractswithrelevantco-factorstodrivegeneexpression. Onceluciferaseisexpressed,itcangeneratelightinanenzymaticassayandtheamountoflightmeasuredispositivelycorrelatedwiththelevelofTFactivation. |
Data:
Analysisofp53PathwayReporterRKO StableCellLineinresponsetostimuli. Thecellswereseededona96-wellplateforovernightwithDMEMincluding10%FBS.Thecellsthenweretreatedwithorwithout2ug/mlQuinacrinerespectivelyinDMEMand0.1%FBSfor16hours.
