Phosphodiesterases(PDEs)playanimportantroleinthedynamicregulationofcAMPandcGMPsignaling.PDE4Dhas3",5"-cyclic-AMPphosphodiesteraseactivityanddegradescAMP.InhibitionofPDE4DactivitybyitsinhibitorsleadstoanelevatedintracellularlevelofcAMP.ThePDE4Dgeneencodesatleast9differentisoforms,andhasbeenlinkedtostroke,asthma,arrhythmia,andcardiacmyopathy,makingitanimportanttherapeutictarget.
ThePDE4DTransientPackisdesignedtoprovidethetoolsnecessaryfortransientlytransfectingandforscreeninginhibitorsofPDE4D7inculturedHEK293cells.
TheassayisbasedontransfectingcellswiththeCREluciferasereporter.CREreportercontainsthefireflyluciferasegeneunderthecontrolofcAMPresponseelement(CRE).ElevationofintracellularcAMPactivatesCREbindingprotein(CREB)tobindCREandinducetheexpressionofluciferase.ForskoliniscommonlyusedtoraisetheintracellularlevelofcAMPincellphysiologystudies.WhencellstransientlytransfectedwithCREreporterareactivatedbyforskolin,theintracellularlevelofcAMPisupregulated,whichinducestheexpressionofCREluciferasereporter.
However,whencellsareco-transfectedwithPDE4D7expressionvectorandCREreporter,thelevelofforskolin-inducedcAMPisreduced,resultinginlowerexpressionlevelofluciferase.WhencellsaretreatedwithPDE4DinhibitortoinhibitPDE4D7activity,cAMPlevelisrestored,resultinginhigherluciferaseactivity.ThePDE4DTransientPackcontainstransfection-readyCREluciferasereporter.Thisreportercontainstransfection-readyvectorsforPDE4D7expressionandaCREvectorcontainingfireflyluciferaseasacAMPpathway-responsivereporterandconstitutivelyexpressingRenillaluciferaseasatransfectioncontrol.
ItalsoincludestheDualLuciferasedetectionreagentstodetectbothluciferaseactivitiesandspecializedmediumforgrowingandassayingHEK293cells.ThekeytothePDE4DCell-BasedActivityReporterDetectionValuepackistheCREluciferasereportervector,whichisacAMP/PKACellSignalingPathway-responsivereporter.ThisreportercontainsthefireflyluciferasegeneunderthecontrolofmultimerizedcAMPresponseelements(CRE)locatedupstreamofaminimalpromoter.Thevectorispremixedwithconstitutively-expressingRenilla(seapansy)luciferasevectorthatservesasaninternalcontrolfortransfectionefficiency.ThepackalsoincludesthePDE4D7expressionvector,andtheadenylatecyclaseactivator,forskolin.Additionally,thepackincludescellculturemedium(BPSMedium1)thathasbeenoptimizedforusewithHEK293andHeLacells*.BPSMedium1includesMEMmedium,10%fetalbovineserum,1%non-essentialaminoacids,sodiumpyruvate,and1%Pen/Strep.
Finally,thepackprovidestheDualLuciferase(Firefly-Renilla)AssaySystem.Theseluciferasereagentsprovidehighlysensitive,stabledetectionoffireflyluciferaseactivityandRenillaluciferaseactivity.Thedualluciferasereagentscanbeuseddirectlyincellsingrowthmedium,andcanbedetectedwithanyluminometer;automatedinjectorsarenotrequired.
*Note:thekitmaybeusedwithothercelllinesthanHEK293orHeLa,butanalternatecellculturemediummayberequiredforoptimalcellgrowth
•MonitorcAMP/PDE4Dsignalingpathwayactivity
2.FanChung,K.(2006)Phosphodiesteraseinhibitorsinairwaysdisease.Eur.J.Pharmacol.533(1-3):110-117
3.Malik,R.etal.(2008)Cloning,stableexpressionofhumanphosphodiesterase7AanddevelopmentofanassayforscreeningofPDE7selectiveinhibitors.Appl.Microbiol.Biotechnol.77(5):1167-1173
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