Description:
CHOPResponsiveLuciferaseReporterMia-Paca2StableCellLineisderivedfromhumanpancreaticcancer,andstablyexpressfireflyluciferasereportergeneunderthecontroloftheUnfoldedProteinResponse,ERstressresponseelement. ThiscelllineisanidealcellularmodelformonitoringtheactivationofUnfoldedProteinResponse,ERstressReceptorSignalingPathwaytriggeredbystimulitreatment,enforcedgeneexpressionandgeneknockdown.Principle:
CHOP,alsoknownasGADD153,isamultifunctionaltranscriptionfactoroftheUnfoldedProteinResponseorendoplasmicreticulumstress(ERstress). DuringUPR,theactivationofPERK-ATF4pathwayupregulatesCHOPproteinlevel,althoughIRE1andATF6pathwayscanalsoregulateCHOPexpression. TranscriptionallyactiveCHOPincreasestargetgenes,suchasEro1andGADD34,andmediatesERstress-inducedapoptosis. CHOPalsoplaysanimportantroleinproteinsynthesis,intracellularcalciumregulation,andoxidationduringERstress.
SignosishasdevelopedCHOPluciferasereporterstablecelllinebytransducingcellswithbaculoviruscontainingbothCHOPluciferasereporterandhygromycinexpressioncassette. Thehygromycinresistantclonesweresubsequentlyscreenedforthapsigargin-inducedluciferaseactivity. ThecelllinecanbeusedasareportersystemformonitoringtheactivityofCHOPtriggeredbystimulitreatment,suchasthapsigargin,tunicamycin,geneoverexpressionandgeneknockdown. Thecellscontainnoviralparticlesandrequirehandlingatbiosafetylevel1protocol.
PrinciplebehindTFluciferasereporter. TFluciferasereporterstablecelllineutilizesartificialpromoterconstructstodriveluciferaseexpression. Thepromoterregioncanconsistsofmultiplerepeatsofacis-elementTFbindingsite,aDNAfragmentfromthepromoterregionofaknownTFdownstreamgene,oraDNAfragmentcontainingputative/knownTFbindingsites. ThereareseveralwaysthataTFcanbeactivated,suchasthroughextracellularstimuliorthroughintracellularsignalingpathways. Onceactivated,theTFtranslocatestothenucleusandofteninteractswithrelevantco-factorstodrivegeneexpression. Onceluciferaseisexpressed,itcangeneratelightinanenzymaticassayandtheamountoflightmeasuredispositivelycorrelatedwiththelevelofTFactivation. |
Data:
AnalysisofSL-0025CHOPreporteractivityinresponsetothapsigargintreatment. TheMia-Paca2cellswereseededona96-wellplateforovernightwithDMEMincluding10%FBS.Thecellsthenweretreatedwithorwithout300nMthapsigargininDMEMand10%FBSfor16hours. Mia-Paca2-CHOPLuciferaseReporterCellLineexhibitsdose-dependentincreaseinluciferaseactivitywhencomparedtountreatedcells.
