TheWnt/β-cateninsignalingpathwaycontrolsalargeanddiversesetofcellfatedecisionsinembryonicdevelopment,adultorganmaintenanceanddisease.Wntproteinsbindtoreceptorsonthecellsurface,initiatingasignalingcascadethatleadstostABIlizationandnucleartranslocationofβ-catenin.β-cateninthenbindstoTCF/LEFtranscriptionfactorsinthenucleus,leADIngtotranscriptionandexpressionofWnt-responsivegenes.
TheTCF/LEFTransientPackisdesignedtoprovidethetoolsnecessaryfortransientlytransfectingandmonitoringtheactivityofWnt/Beta-cateninsignalingpathwayintheculturedHEK293cells.TheTCF/LEFTransientPackcontainstransfection-readyvectorscontainingfireflyluciferaseasaWntpathway-responsivereporterandconstitutivelyexpressingRenillaluciferaseasatransfectioncontrol. ItalsoincludestheDualLuciferasedetectionreagentstodetectbothluciferaseactivitiesandspecializedmediumforgrowingandassayingHEK293cells.
ThekeytotheTCF/LEFTransientPackistheTCF/LEFluciferasereportervector,whichisaWntpathway-responsivereporter.ThisreportercontainsafireflyluciferasegeneunderthecontrolofmultimerizedTCF/LEFresponsiveelementlocatedupstreamofaminimalpromoter.TheTCF/LEFreporterispremixedwithconstitutivelyexpressingRenillaluciferasevectorthatservesasinternalcontrolfortransfectionefficiency.
Thepackalsoincludesanon-inducIBLefireflyluciferasevectorpremixedwithconstitutivelyexpressingRenillaluciferasevectorasnegativecontrol.Thenon-inducibleluciferasevectorcontainsafireflyluciferasegeneunderthecontrolofaminimalpromoter,withoutanyadditionalresponseelements.Thenegativecontroliscriticaltodeterminingpathwayspecificeffectsandbackgroundluciferaseactivity.
Additionally,thepackincludescellculturemedium(BPSMedium1)thathasbeenoptimizedforusewithHEK293cells*.BPSMedium1includesMEMmedium,10%fetalbovineserum,1%non-essentialaminoacids,sodiumpyruvate,and1%Pen/Strep. Finally,thepackprovidestheDualLuciferase(Firefly-Renilla)AssaySystem.Theseluciferasereagentsprovidehighlysensitive,stabledetectionoffireflyluciferaseactivityandRenillaluciferaseactivity. Thedualluciferasereagentscanbeuseddirectlyincellsingrowthmedium,andcanbedetectedwithanyluminometer;automatedinjectorsarenotrequired.
*Note: thekitmaybeusedwithothercelllinesthanHEK293,butanalternatecellculturemediummayberequiredforoptimalcellgrowth.
•ScreenactivatorsorinhibitorsofWnt/β-cateninsignalingpathway.
•StudyeffectsofRNAiorgeneoverexpressionontheactivityofWntpathway.