TheMycsignalingpathwayplaysanimportantroleincellproliferation,differentiation,transformationandapoptosis.Thec-MycproteinisatranscriptionfactorthatheterodimerizeswithMaxtoregulateMycsignalingpathwayresponsivegenes.Mycmutationshavebeenlinkedtothedevelopmentofanumberofhumancancers,includingBurkitt’slymphoma,cervical,ovarian,breast,lungandpancreaticcarcinoma,makingMycapromisingtherapeutictargetforcancertreatment.
TheMycTransientPackisdesignedtoprovidethetoolsnecessaryfortransientlytransfectingandmonitoringtheactivityoftheMycsignalingpathwayinculturedHCT116cells.TheMycTransientPackcontainstransfection-readyvectorscontainingfireflyluciferaseasaMycpathway-responsivereporterandconstitutivelyexpressingRenillaluciferaseasatransfectioncontrol. ItalsoincludestheDualLuciferasedetectionreagentstodetectbothluciferaseactivitiesandspecializedmediumforgrowingandassayingHCT116cells.
.ThekeytotheMycTransientPackisthe expressionvectorsforc-MycandMycluciferasereportervector.Insidethecells,c-MycwillbindtoMax,translocatetothenucleus,andinduceexpressionoftheMycluciferasereportervector.ThisreportercontainsthefireflyluciferasegeneunderthecontrolofmultimerizedMycresponsiveelementslocatedupstreamofaminimalpromoter.TheMycreporterispremixedwithconstitutively-expressingRenilla(seapansy)luciferasevector,whichservesasaninternalpositivecontrolfortransfectionefficiency.
Thepackalsoincludesanon-inducIBLefireflyluciferasevectorpremixedwithconstitutively-expressingRenillaluciferasevectorasanegativecontrol.Thenon-inducibleluciferasevectorcontainsafireflyluciferasegeneunderthecontrolofaminimalpromoter,butwithoutanyadditionalresponseelements.Thisnegativecontroliscriticalfordeterminingpathway-specificeffectsandbackgroundluciferaseactivity.An“empty”expressionvectorwithoutthec-Mycgeneisalsoprovidedasanadditionalnegativecontrol.
Additionally,thepackincludescellculturemediumthathasbeenoptimizedforusewithHCT116cells*. HCT116isahumancoloncancercelllinewithamutatedβ-cateninwhichleadstotheaccumulationofβ-cateninandconstitutiveactivationoftheMycsignalingpathway. Thismediumincludes10%fetalbovineserum,and1%Pen/Strep. Finally,thepackprovidestheDualLuciferase(Firefly-Renilla)AssaySystem.Thesereagentsprovidehighlysensitive,stabledetectionoffireflyluciferaseactivityandRenillaluciferaseactivity. Thedualluciferasereagentscanbeuseddirectlyincellsingrowthmedium,andcanbedetectedwithanyluminometer;automatedinjectorsarenotrequired.
*Note: thekitmaybeusedwithothercelllinesthanHCT116,butanalternatecellculturemediummayberequiredforoptimalcellgrowth. ForHEK293cells,werecommendusingBPSMedium1(BPSBioscience,#79259).
•ScreenactivatorsorinhibitorsoftheMycsignalingpathway.
•StudyeffectsofRNAiorgeneoverexpressionontheactivityoftheMycpathway.
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