Theglucocorticoidsignalingpathwayplaysanimportantroleindevelopment,fluidhomeostasis,cognition,immuneresponseandmetabolism.Glucocorticoidsareaclassofsteroidhormonesthatbindtotheglucocorticoidreceptor,causingittotranslocatetothenucleus.Upontranslocation,thereceptorcanregulatethetranscriptionofalargenumberofgenes,includingthosethatregulateglucosemetabolismandinflammatoryresponses.
TheGAL4TransientPackisdesignedtoprovidethetoolsnecessaryfortransientlytransfectingandmonitoringtheactivityoftheglucocorticoidsignalingpathwayinculturedHEK293cells.Thekitcontainstransfection-readyvectorscontainingfireflyluciferaseasaglucocorticoidpathway-responsivereporterandconstitutivelyexpressingRenillaluciferaseasatransfectioncontrol. ItalsoincludestheDualLuciferasedetectionreagentstodetectbothluciferaseactivitiesandspecializedmediumforgrowingandassayingHEK293cells.
ThekeytotheGAL4TransientPackistheexpressionvectorfortheglucocorticoidreceptorligandbindingdomainthatisfusedtotheDNAbindingdomain(DBD)ofGAL4(GAL4DBD-GR).ThisfusionconstructactivatesfireflyluciferaseexpressionunderthecontrolofamultimerizedGAL4upstreamactivationsequence(UAS).Thisallowsforspecificdetectionofglucocorticoid-inducedactivationoftheglucocorticoidreceptorwithouttheneedforindividualtranscriptionaltargetsandwithlowcross-reactivityforothernuclearreceptorpathways.TheGAL4/UASreporterispremixedwithconstitutivelyexpressingRenilla(seapansy)luciferasevector,whichservesasaninternalpositivecontrolfortransfectionefficiency.
Thepackalsoincludesanon-inducIBLefireflyluciferasevectorpremixedwithconstitutively-expressingRenillaluciferasevectorasanegativecontrol.Thenon-inducibleluciferasevectorcontainsafireflyluciferasegeneunderthecontrolofaminimalpromoter,butwithoutanyadditionalresponseelements.Thenegativecontroliscriticalfordeterminingpathway-specificeffectsandbackgroundluciferaseactivity.
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.
