tet-on-and-tet-off-系统

Tet-Off®andTet-On®GeneExpressionSystemsUserManualPT3001-1(100912)PublishedOctober2012UnitedStates/Canada800.662.2566AsiaPacific+1.650.919.7300Europe+33.(0)1.3904.6880Japan+81.(0)77.543.6116ClontechLaboratories,Inc.ATakaraBioCompany1290TerraBellaAve.MountainView,CA94043TechnicalSupport(US)E-mail:[e-mail]:pBI,VP16andpTet-tTSVectors10H.ViralTetExpression10II.ProtocolOverview11III.ListofComponents13IV.AdditionalMaterialsRequired14V.PlasmidManipulations17A.PropagationofVectorPlasmids17B.GeneratingyourGene-SpecificExpressionVector17VI.CellCultureGuidelines18A.GeneralInformation18B.CharacteristicsofTet-OffandTet-OnCellLines18C.StartingTetCellCulturesfromFrozenStocks18D.PreparingFrozenStocksofTetCellLines19VII.PilotExperiments20A.PilotExperimentwiththeCHO-AA8-LucTet-OfforU2-OS-LucTet-OnControlCellLine20B.TitratingG418,Hygromycin,andPuromycin(KillCurves)21C.TestPotentialHostCellsbyTransientTransfectionwithpTRE2hyg-LucandpTet-OfforpTet-On23VIII.DevelopmentofStableCellLines24A.TransfectionandSelectionofStableCellLines24B.ScreeningStableCellLines26IX.DevelopmentofDouble-StableCellLines27A.TestpTRE-GeneXbyTransientTransfectionintoaTet-OfforTet-OnCellLine27B.StablyTransfectandSelectDouble-StableCellLines27C.StablyTransfectandSelectDouble-StableCellLines—Cotransfection29D.ScreeningDouble-StableCellLines30E.WorkingwithDouble-StableCellLines30ProtocolNo.PT3001-1:VectorInformation34AppendixB:Glossary46ListofFiguresFigure1.Inducibleon/offcontrolofgeneexpressionintheTetSystems4Figure2.SchematicofgeneregulationintheTetSystems6Figure3.LuciferaseexpressionisrapidlyinducedinaTet-OffcelllineinresponsetoremovalofDox.8Figure4.DevelopingTet-OffandTet-OnCellLines12Figure5.FoldinductionofluciferaseactivityindifferentlotsofFBS20Figure6.Dose-responsecurvesfortheTetControlCellLines22Figure7.FlowchartfordevelopingTetCellLines25Figure8.Flowchartfordevelopingdouble-stableTetCellLines28Figure9.pTet-OffandpTet-Oncompositevectormap37Figure10.pTRE2hygandpTRE2purplasmidmapandMCS38Figure11.pTRE-TightvectormapandMCS39Figure12.pTRE-Myc,-HAand-6xHNcompositevectormapandMCS40Figure13.pTRE2Marker-Myc,-HA,and-6xHNcompositevectormapandMCS41Figure14.pTK-Hygplasmidmap42Figure15.ThepBIexpressioncassette43Figure16.VP16MinimalDomainvectors44Figure17.ControlledexpressioninacelllinecoexpressingtTSandrtTA45ListofTablesTableI.Tet-OffandTet-OnVectorAlignment34TableII.TetSystemsVectorInformation35ClontechLaboratories,Inc.–GAPDH–Tet-OffTet-On–+–+I.IntroductionFigure1.Inducibleon/offcontrolofgeneexpressionintheTetSystems.PanelA.Double-stablecelllinesweredevelopedbystablytransfectingHeLaTet-OfforHeLaTet-OncellswithaplasmidcontainingE.colilacZundercontroloftheTetresponseelement(TRE).Cellswerecultured+/–1µg/mlDox.ForNorthernanalysis,10µgoftotalRNAwasloadedperlane,andtheblotwashybridizedsimultaneouslywithprobestolacZandtheGAPDHhousekeepinggene(Gossenetal.,1995;reprintedwithpermissionoftheauthor).PanelB.HeLaS3Tet-OffcellswerestablytransfectedwithaplasmidexpressingBcl-2undercontroloftheTREandgrowninthepresenceoftheindicatedamountsofTc.AWesternblotcontaining100µgoftotalproteinfromeachconditionwasprobedwithhumanBcl-2-specificandhumancyclin-B1-specificmousemonoclonalantibodies.Basedonscanningdensitometry,removalofTcgave~100-foldinductionofBcl-2.Fordetails,seeYin&Schimke(1995).A.SummaryTheTet-OffandTet-OnGeneExpressionSystemsandthepremadeTet-OffandTet-OnCellLinesgiveresearchersreadyaccesstotheregulated,high-levelgeneexpressionsystemsdescribedbyGossen&Bujard(1992;Tet-Off)andGossenetal.(1995;Tet-On).IntheTet-Offsystem,geneexpressionisturnedonwhentetracycline(Tc)ordoxycycline(Dox;aTcderivative)isremovedfromtheculturemedium.Incontrast,expressionisturnedonintheTet-OnsystembytheadditionofDox(Figure1A).TheTet-OnsystemisresponsiveonlytoDox,nottoTc.BothsystemspermitgeneexpressiontobetightlyregulatedinresponsetovaryingconcentrationsofTcorDox(Figure1B).MaximalexpressionlevelsinTetsystemsareveryhighandcomparefavorablywiththemaximallevelsobtainablefromstrong,constitutivemammalianpromoterssuchasCMV(Yinetal.,1996).Unlikeotherinduciblemammalianexpressionsystems,generegulationintheTetSystemsishighlyspecific,sointerpretationofresultsisnotcomplicatedbypleiotropiceffectsornonspecificinduction.BA2,00064210.50.250Tc(ng/ml):Cyclin–Bcl-2–ProtocolNo.PT3001-1