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Tet-Off® and Tet-On®
Gene Expression SystemsUser Manual
Cat. No. 630921 630922 PT3001-1 (PR58969)Published 13 September 2005
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3001-1 � Version No. PR58969
Tet Systems User Manual
Table of Contents
I. Introduction 4
A. Summary 4 B. TheTet-OffandTet-OnSystems 5 C. AdvantagesoftheTetSystems 7 D. Tet-Offvs.Tet-OnSystems 9 E. Tetracyclinevs.Doxycycline 9 F. AdditionalTetResponseVectors 10 G. BeyondtheBasics:pBI,VP16andpTet-tTSVectors 11 H. RetroviralTetExpression 11 I. AdenoviralTetExpression 11
II. Protocol Overview 12
III. List of Components 14
IV. Additional Materials Required 15
V. Plasmid Manipulations 18
A. PropagationofVectorPlasmids 18 B. GeneratingyourGene-SpecificExpressionVector 18 VI. Cell Culture Guidelines 19
A. GeneralInformation 19 B. CharacteristicsofTet-OffandTet-OnCellLines 19 C. StartingTetCellCulturesfromFrozenStocks 19 D. PreparingFrozenStocksofTetCellLines 20 VII. Pilot Experiments 21
A. PilotExperimentwiththeCHO-AA8-LucTet-OfforU2-OS-Luc Tet-OnControlCellLine 21
B. TitratingG418,Hygromycin,andPuromycin(KillCurves) 22
C. TestPotentialHostCellsbyTransientTransfectionwithpTRE2hyg-LucandpTet-OfforpTet-On 24
VIII. Development of Stable Cell Lines 25
A. TransfectionandSelectionofStableCellLines 25 B. ScreeningStableCellLines 27
IX. Development of Double-Stable Cell Lines 28
A. TestpTRE-GeneXbyTransientTransfectionintoaTet-OfforTet-OnCellLine 28
B. StablyTransfectandSelectDouble-StableCellLines 28 C. StablyTransfectandSelectDouble-StableCellLines— Cotransfection 30
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Table of Contents continued
D. ScreeningDouble-StableCellLines 31 E. WorkingwithDouble-StableCellLines 31
X. References 33
XI. Related Products 35
Appendix A: Vector Information 37Appendix B: Glossary 51
List of Figures
Figure1. Inducibleon/offcontrolofgeneexpressionintheTetSystems 4Figure2. SchematicofgeneregulationintheTetSystems 6Figure3. LuciferaseexpressionisrapidlyinducedinaTet-Offcelllinein
responsetoremovalofDox. 8Figure4. DevelopingTet-OffandTet-OnCellLines 13Figure5. FoldinductionofluciferaseactivityindifferentlotsofFBS 21Figure6. Dose-responsecurvesfortheTetControlCellLines 23Figure7. FlowchartfordevelopingTetCellLines 26Figure8. Flowchartfordevelopingdouble-stableTetCellLines 29Figure9. pTet-OffandpTet-Oncompositevectormap 42Figure10. pTRE2hygandpTRE2purplasmidmapandMCS 43Figure11. pTRE-TightvectormapandMCS 44Figure12. pTRE-Myc,-HAand-6xHNcompositevectormapandMCS 45Figure13. pTRE2Marker-Myc,-HA,and-6xHN
compositevectormapandMCS 46Figure14. pTK-Hygplasmidmap 47Figure15. ThepBIexpressioncassette 48Figure16. VP16MinimalDomainvectors 49Figure17. Controlledexpressioninacelllinecoexpressing
tTSandrtTA 50
List of TablesTableI. Tet-OffandTet-OnVectorAlignment 37
TableII. TetSystemsVectorInformation 39
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3001-1 � Version No. PR58969
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D60167pr.bw/pTet-OffDRAFT3/1/96
Hind III(2250)
Xho I(2)
pTet-OffpTet-On
�.� kb
Col E1ori
Ampr
PCMV
SV40poly A
Neor
Xho I(4461)
Bsa I(3546)
Sca I(3949)
VP16AD
(r)tetR
Hind III (pTet-On only)(871)
(r)tTA
= Mutations that convert TetR to rTetR (and tTA to rtTA)
Dox:
LacZ –
GAPDH –
Tet-Off Tet-On – + – +
I. Introduction
Figure 1. Inducible on/off control of gene expression in the Tet Systems. Panel A. Double-stablecelllinesweredevelopedbystablytransfectingHeLaTet-OfforHeLaTet-OncellswithaplasmidcontainingE. coli lacZundercontroloftheTetresponseelement(TRE).Cellswerecultured+/–1µg/mlDox.ForNorthernanalysis,10µgoftotalRNAwasloadedperlane,andtheblotwashybridizedsimultaneouslywithprobestolacZandtheGAPDHhousekeepinggene(Gossenet al.,1995;reprintedwithpermissionoftheauthor).Panel B.HeLaS3Tet-OffcellswerestablytransfectedwithaplasmidexpressingBcl-2undercontroloftheTREandgrowninthepresenceoftheindicatedamountsofTc.AWesternblotcontaining100µgoftotalproteinfromeachconditionwasprobedwithhumanBcl-2-specificandhumancyclin-B1-specificmousemonoclonalantibodies.Basedonscanningdensitometry,removalofTcgave~100-foldinductionofBcl-2.Fordetails,seeYin&Schimke(1995).
A. Summary The Tet-Off®and Tet-On® Gene Expression Systemsandthepremade
Tet-Offand Tet-On Cell Linesgiveresearchersreadyaccesstotheregulated,high-levelgeneexpressionsystemsdescribedbyGossen&Bujard(1992;Tet-Off)andGossenet al. (1995;Tet-On). In theTet-Off system,geneexpressionisturnedonwhentetracycline(Tc)ordoxycycline(Dox;aTcderivative)isremovedfromtheculturemedium.Incontrast,expressionisturnedonintheTet-OnsystembytheadditionofDox(Figure1A).TheTet-OnsystemisresponsiveonlytoDox,nottoTc.BothsystemspermitgeneexpressiontobetightlyregulatedinresponsetovaryingconcentrationsofTcorDox(Figure1B).
Maximal expression levels in Tet systems are very high and comparefavorably with the maximal levels obtainable from strong, constitutivemammalianpromoterssuchasCMV(Yinet al. ,1996).Unlikeotherinduciblemammalian expression systems, gene regulation in theTet Systems ishighlyspecific,sointerpretationofresultsisnotcomplicatedbypleiotropiceffectsornonspecificinduction.
BA
2,000 6 4 2 1 0.5 0.25 0 Tc (ng/ml):
Cyclin –
Bcl-2 –
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I. Introduction continued
SeeAppendixAortheVectorInformationPacketsprovidedformapsanddetailedinformationontheTetSystemVectors.ForacompletelistofTetSystemsreferences,visitourwebsiteatwww.clontech.com.
B. The Tet-Off and Tet-On Systems InE. coli,theTetrepressorprotein(TetR)negativelyregulatesthegenes
ofthetetracycline-resistanceoperonontheTn10transposon.TetRblockstranscriptionofthesegenesbybindingtothetetoperatorsequences(tetO)in theabsenceofTc.TetRand tetOprovidethebasisofregulationandinductionforuseinmammalianexperimentalsystems.
ThefirstcriticalcomponentoftheTetSystemsisthe regulatory protein, basedonTetR.IntheTet-OffSystem,this37-kDaprotein isafusionofaminoacids1–207ofTetRandtheC-terminal127a.a.oftheHerpessimplexvirusVP16activationdomain(AD;Triezenberget al. ,1988).AdditionoftheVP16domainconvertstheTetRfromatranscriptionalrepressortoatranscriptionalactivator,andtheresultinghybridproteinisknownasthetetracycline-controlledtransactivator(tTA).tTAisencodedbythepTet-Offregulatorplasmid,whichalsoincludesaneomycin-resistancegenetopermitselectionofstablytransfectedcells.
TheTet-OnsystemissimilartotheTet-Offsystem,buttheregulatoryproteinisbasedona "reverse"Tet repressor (rTetR)whichwascreatedby fouraminoacidchangesinTetR(Hillen&Berens,1994;Gossenet al. ,1995).Theresultingprotein,rtTA(reversetTA),isencodedbythepTet-Onregulatorplasmid,whichalsocontainsaneomycin-resistancegene.
Thesecondcriticalcomponentistheresponse plasmidwhichexpressesagene of interest (Gene X)undercontrolof the tetracycline-responseelement,orTRE.WeprovidetworesponsevectorseriesfortheTetSystems.Ouroriginalvectorseries—pTREoritsvariants—containtheTRE,whichconsistsofsevendirectrepeatsofa42-bpsequencecontainingthetetO,located just upstream of the minimal CMV promoter (PminCMV). PminCMVlacks the strong enhancer elements normally associated with the CMVimmediateearlypromoter.Becausetheseenhancerelementsaremissing,thereisextremelylowbackgroundexpressionofGeneXfromtheTREintheabsenceofbindingbytheTetRdomainoftTAortherTetRdomainofrtTA.Oursecondresponsevectorseries—pTRE-Tight—containamodifiedTRE(TREmod)upstreamofanalteredminimalCMVpromoter(PminCMV∆),resultinginfurtherreducedbasalexpressionofGeneX.pTRE-Tightcanfullyminimizebackgroundexpressionincertaincelllines,andisespeciallyusefulincaseswherebackgroundexpressionisunacceptable,suchastheexpressionofproteinsthatareextremelypotentortoxictothehostcell(April2003Clontechniques).
TheultimategoalinsettingupafunctionalTetSystemiscreatingadouble-stable Tet cell line which contains both the regulatory and responseplasmids. When cells contain both the regulatory (pTet-Off or pTet-On)
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I. Introduction continued
Figure 2. Schematic of gene regulation in the Tet-Off and Tet-On Systems. Tet-Off:TheTREislocatedupstreamoftheminimalimmediateearlypromoterofcytomegalovirus(PminCMV),whichissilentintheabsenceofactivation.tTAbindstheTRE—andtherebyactivatestranscriptionofGeneX—intheabsenceofTcorDox. Tet-On:The"reverse"Tetrepressor(rTetR)wascreatedbyfouraminoacidchangesthatreversetheprotein’sresponsetoDox.Asaresultofthesechanges,therTetRdomainofrtTAbindstheTREandactivatestranscriptioninthepresenceofDox.PleaseseeAppendixAformapsanddetailedvectorinformation.
Tet-On
Tet-Off
Transcription
Gene of interestPminCMVTRE
X
rtTA
VP1�rtetRPCMV
Transcription
ADDDOX
REMOVEDOX
Tet-On System
rtTA ( )binds TRE and activates transcription in the presence of Dox
Gene of interestPminCMVTRE
TranscriptionXTranscription
tetR
tTA
PCMV VP1�
ADDDOX
REMOVEDOX
tTA ( )binds TRE and activates transcription in the absence of Dox
Tet-Off System
Gene of interestPminCMVTRE Gene of interestPminCMVTRE
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I. Introduction continued
andtheresponse(e.g.pTRE-GeneX)Vectors,GeneXisonlyexpresseduponbindingofthetTAorrtTAproteintotheTRE(Figure2).IntheTet-OffSystem,tTAbindstheTREandactivatestranscriptionintheabsence ofTcorDox.IntheTet-OnSystem,rtTAbindstheTREandactivatestranscriptioninthepresenceofDox.InbothTet-OnandTet-OffSystems,transcriptionisturnedonoroffinresponsetoDoxinapreciseanddose-dependentmanner.
You can greatly reduce the time needed to establish aTet cell line bypurchasing one of our premade Tet Cell Lines, which already stablyexpresstheappropriateregulatoryprotein.AlistofavailableTet-OffandTet-On Cell Lines is available from our Tet Systems product page atwww.clontech.com.
Notethatadditionofanuclearlocalizationsequence[nls]totTAorrtTAalters the protein’s regulatory function (M. Gossen & H. Bujard, pers.comm.).AdditionofannlstotTAorrtTAincreasesmaximumexpressionbutalsoincreasesbackgroundexpressionduetoalteredbindingaffinitytotetOsequences(unpublishedobservations).Therefore,werecommendthatyoudonotaddanlstoeithertTAorrtTAforcreatingstableTetcelllines.
C. Advantages of the Tet Systems The Tet-Off and Tet-On systems have several advantages over other
regulatedgeneexpressionsystemsthatfunctioninmammaliancells: • Extremely tight on/off regulation. Background, or leaky, expression
ofGeneX in theabsenceof induction isextremely lowwithpTREoritsvariants(Figure1).Forthelowestbackgroundexpression,usepTRE-TightVectors.
• No pleiotropic effects. When introduced into mammalian cells, theprokaryoticregulatoryproteins(TetRorrTetR,theprokaryoticprecursorsto tTA and rtTA) act very specifically on their target sequences,presumablybecausetheseregulatoryDNAsequencesarenonexistentineukaryoticgenomes(Harkinet al.,1999).
• Highinducibilityandfastresponsetimes.WiththeTetSystems,inductioncanbedetectedwithin30minutes(Figure3)usingnontoxiclevelsofinducer.Inductionlevelsupto10,000-foldhavebeenobserved(resultsnotshown).Incontrast,othersystemsformammalianexpressionexhibitslowinduction(uptoseveraldays),incompleteinduction(comparedtorepressor-freecontrols),lowoverallinduction(oftennomorethan100-fold),andhigh(nearlycytotoxic) levelsof inducer(reviewedbyGossenet al.,1993;Yarronton,1992).
• High absolute expression levels. Maximal expression levels in theTet systems can be higher than expression levels obtained fromthe CMV promoter or other constitutive promoters. For example,
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I. Introduction continued
Yinet al. ,(1996)reportedthatthemaximallevelofluciferaseexpressioninHeLaTet-OffcellstransientlytransfectedwithpTRE-Lucis35-foldhigherthanthatobtainedwithHeLacellstransientlytransfectedwithaplasmidexpressingluciferasefromthewild-typeCMVpromoter.
• Well-characterized inducer. In contrast to the inducerused inothersystems,suchasintheecdysonesystem,TcandDoxareinexpensive,wellcharacterized,andyieldhighlyreproducibleresults.
• Activationofapromoter,ratherthanrepression,tocontrolexpression.Tocompletelyshutofftranscription,repression-basedsystemsrequireveryhigh—anddifficulttoattain—levelsofrepressortoensure100%occupancyoftheregulatorysites.Evenifsuitablyhighlevelsofrepressorcanbeobtained,thepresenceofhighrepressorlevelsmakesitdifficulttoachieverapid,high-level induction(Yaoet al.,1998).Foramorecompletediscussionoftheadvantagesofactivationversusrepression,seeGossenet al.,(1993).
IncontrasttotheheterologousTetSystems,homologoussystemsbasedoneukaryoticregulatoryelementsaresubjecttooneormoreofthefollowingproblems:
• Inducingstimulusispleiotropic,i.e.,thegeneofinterestisnottheonlygeneaffectedbytheinducingstimulus.
Time (hr)
1 2 3 54 6 7 8 90
Luci
fera
se a
ctiv
ity (R
LU; x
103
)
5
10
15
20
25
30
0
removal of 1 �g/ml Dox
addition of 1 �g/ml Dox
Figure 3. Luciferase expression is rapidly induced in a Tet-Off cell line in response to removal of Dox. TheCHO-K1-EGFP-LucTet-Offcontrolcell lineexpressesthetTAandcontainsastablyintegrated copy of the firefly luciferase gene under control of the TRE. Luciferase activity wascontinuouslymonitoredwithafluorescentimagingplatereader(FLIPR,MolecularDevicesCorp.)afteradditionorremovalof1µg/mlDoxfromtheculturemedium(Cunninghametal.,1997).
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I. Introduction continued
• Itcanbeverydifficulttodistinguishspecificfromnonspecificeventsinanexpressionsystembasedonhomologousregulatoryelements.Thisislargelyduetothemodularnatureofeukaryoticpromoterswhichinteractwithavarietyoftranscriptionfactorsthatare,inturn,involvedintheregulationofmanypromotersand/orenhancers.
• Mostof thecommonlyusedeukaryoticpromotersaretoo“leaky” tomaintainthegeneofinterestinthefullyrepressed(“off”)state,limitingtheirusefulnessforexpressingtoxicproteins.
• Maximallevelofinductionisusuallynotveryhigh. Thus,ofthesystemsdescribedtodate,onlytheTetSystemsexhibittighton/off
regulation,absenceofpleiotropiceffects,highinductionlevels,highabsoluteexpression,andrapidinductiontimes(Gossenet al.,1993;1994).
D. Tet-Off vs. Tet-On Systems AlthoughtheTet-Offsystemhasbeenstudiedmoreextensivelythanthe
Tet-Onsystem,thetwosystemsaretrulycomplementary.Whenproperlyoptimized, both systems give tight on/off control of gene expression,regulateddose-dependentinductionwithsimilarkineticsofinduction,andhighabsolutelevelsofgeneexpression.Thus,formostpurposes,thereisnoinherentadvantageofusingonesystemovertheother.
WiththeTet-Offsystem,itisnecessarytokeepTcorDoxinthemediumtomaintainthenative(off)state.BecauseTcandDoxhaverelativelyshorthalf-lives(seebelow),youmustaddTcorDoxtothemediumatleastevery48hours to suppressexpressionofGeneX.Conversely, in theTet-Onsystem,thenative(off)stateismaintaineduntilinduction.Forthisreason,Tet-Onmaybemoreconvenientintransgenicapplications,becauseyouneedonlyaddDoxtotheanimals'dietwheninductionisdesired.
E. Tetracycline vs. Doxycycline TheTet-OnSystemisonlyresponsivetoDox,notTc(Gossen&Bujard,
1995). In contrast,Tet-Off systems respondequallywell to eitherTcorDox.We recommend thatyouuseDox forallTetSystemexperiments,inpartbecauseasignificantlylowerconcentrationofDoxisrequiredforcompleteactivationorinactivation(0.01–1µg/mlDoxvs.1–2µg/mlTc).Inbothsystems,theantibioticsareusedatconcentrationsfarbelowcytotoxiclevelsforeithercellcultureortransgenicstudies.Inaddition,Doxhasalongerhalf-life(24hours)thanTc(12hours).Thus,fortheTet-OffSystem,youmayprefertouseDoxforlong-termmaintenanceofantibioticlevelsandswitchtoTcinpreparationforinduction.
OtherTcderivativeshavebeenusedsuccessfullyas the inducer inTetsystems(Gossen&Bujard,1993).AffinityforTetRandantibioticpotencyareapparentlymediatedbydifferentchemicalmoieties;somederivatives,suchasanhydrotetracycline,haveanincreasedaffinityforTetRanddecreasedantibioticactivity(Gossenet al. ,1993).
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F. Additional Tet Response Vectors ThecompleteTet-OffandTet-OnGeneExpressionSystemsareprovided
withpTRE2hygastheresponsevector.InadditiontotheTREregulatoryelementandamultiplecloningsite,thisvectoralsoexpressesthehygromycinresistancegene,permittingeasyselectionofstabletransfectants.WealsoofferpTRE2pur(Cat.No.631013)foranalternativeselectionschemeusingpuromycin.
Another response vector, the pTRE-Tight Vector (Cat. No. 631059), isavailableseparately.pTRE-TightcontainsamodifiedTREelement(TREmod)thatcanminimizebasalexpressionincertaincelllines.Thisvectorisalsoofferedinareporterformat,thepTRE-Tight-DsRed2Vector(Cat.No.631061)expressesavariantofouroriginalredfluorescentprotein.Thisvectordoesnotcontainaselectablegeneandforbestresultsshouldbecotransfectedwith our Linear Selection Marker for hygromycin (Cat. No. 631625) orpuromycin(Cat.No.631626)resistance(April2003Clontechniques).
Additionally, response vectors are available that express your proteinwith a tag to aid in detection and protein purification. These vectorsprovide a way to screen colonies directly for protein expression byWestern analysis using readily available antibodies.These Vectors areavailablewithorwithoutamammalianselectionmarker.pTRE-MycVector(Cat.No.631010),pTRE2hyg2-Myc(Cat.No.631052),andpTRE2pur-Myc(Cat.No.631055)encodeac-Myctag,whichisincorporatedattheN-terminusoftheexpressedprotein.ThepTRE-HA(Cat.No.631012),pTRE2hyg2-HA(Cat.No.631051),andpTRE2pur-HA(Cat.No.631054)Vectorsencodean HA (hemagglutinin) epitope tag at the N-terminus of the expressedprotein, allowing detection of the protein with anti-HA antibodies. ThepTRE-6xHN(Cat.No.631009),pTRE2hyg2-6xHN(Cat.No.631053),andpTRE2pur-6xHN(Cat.No.631056)VectorsexpressproteinsthatarefusedwithsixHis-AsnrepeatsandalloweasypurificationofyourproteinusingTALON®Resinoranyotherimmobilizedmetalaffinitycolumn.
For users of our Creator™ Gene Cloning and Expression System,pLP-TRE2Acceptor Vector allows you to quickly transfer your gene ofinterest into the Tet Systems. pLP-TRE2 must be cotransfected witha Linear Selection Marker to create stable lines. For more informationontheCreatorSystem,pleasevisitourCreatorproduct familypageatwww.clontech.com.
I. Introduction continued
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I. Introduction continued
The pTRE2 Sequencing/PCR Primers (Cat. No. 631103) can be used(5'or3')tosequencejunctionsbetweenaninsertandanyoftheTetsystemresponsevectors.TheycanalsobeusedtoamplifyorconfirmthepresenceofinsertsviaPCRwhentheexpectedinsertsizeislessthan2–3kb.
SeeAppendixAforadditionalinformationonthesevectors.
G. Beyond the Basics: pBI, VP16, and pTet-tTS Vectors TheBidirectional(pBI)TetVectorsarespeciallydesignedresponsevectors
thatallowcoregulatedexpressionoftwogenesundercontrolofasingleTRE.Theyareidealresponsevectorstouseifyoudonothaveafunctionalassayforyourgeneofinterest,becauseyoucanselectforexpressionofthecoregulatedmarkergene,eitherβ-galactosidaseorluciferase.ThesevectorsdonotcontainaselectablegeneandshouldbecotransfectedwithoneoftheLinearSelectionMarkers,pTK-Hyg(Cat.No.631625)orpPUR(Cat.No.631626).
TheVP16MinimalDomainVectors(Cat.No.631019)containaproteinfusionofTetRfusedtoalteredVP16activationdomains.Theseproteinsaretoleratedathigher intracellular levelsandhaveabettertransfectionefficiencyinsomecelltypes.Theyareespeciallyusefulintransgenicstudies,wherehighexpressionofunmodifiedVP16maybetoxictocells.
ThepTet-tTSVector(Cat.No.631011)isdesignedtopreventunregulated("leaky")geneexpressionintheTet-OnSystem.Itencodesatranscriptionalsilencer(tTS)thatblockstranscriptionofgenesundercontroloftheTREintheabsenceofDox.pTet-tTSisidealforregulatedexpressionoftoxicgenesorotherapplicationsthatrequireextremelylowlevelbasalexpression.(Alternatively,use thepTRE-Tight responseplasmid [Cat.No.631059],whichcontainsamodifiedTREelementthatcanminimizebasalexpressionincertaincelllines).
SeeAppendixAforadditionalinformationonthesevectors.
H. Retroviral Tet Expression TheTetSystemsalsocomeinaretroviralformat.TheRevTet™System
allowsyoutostablyintroducetheelementsoftheTetSystemintovirtuallyanymitoticallyactivecellwithhighefficiency.Formoreinformation,visittheTetSystemsproductpageatwww.clontech.comtodownloadacopyoftheRevTetUserManual(PT3223-1).
I. Adenoviral Tet Expression TheTetgeneexpressionsystemisalsoavailableinanadenoviralversion.
Adeno-X™ Tet-Off and Adeno-X™ Tet-On Expression Systems utilizeadenoviralgenetransfertoinfectdividingandnondividingmammaliancellsfortransient,regulatedexpression.Forfurtherdetails,visitourTetSystemsproduct page at www.clontech.com to obtain a copy of theAdeno-XTet-OffandTet-OnUserManual(PT3496-1).
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II. Protocol Overview
Figure4providesanoverviewforcreatingdouble-stableTet-OfforTet-Oncelllineswhichcontainintegratedcopiesoftheregulatoryandresponsevector—theultimategoal inestablishingtheTetSystem.Formoredetailedflowchartsofeachof the transfectionproceduresseeFigure7 (SectionVIII)andFigure8(SectionIX).IfyouhavepurchasedapremadeTet-OfforTet-OnCellLinefromClontech,youneedonlyperformthesecondtransfectionwithyourpTRE-GeneXconstruct.Important note on simultaneous versus consecutive transfections Ingeneral,werecommendthatyoudonotattempttosavetimebycotransfectingtheregulatorandresponseplasmids.Cotransfectedplasmidstendtocointegrateinto the chromosome, and enhancer elements from the CMV promoter onthe regulator plasmid (pTet-Off or pTet-On) can induce basal expression ofGeneX.Furthermore,cotransfectionpreventscomparisonofmultipleclones,sincedifferencesininductionorabsoluteexpressioncouldbeduetoclone-to-clonevariationintTAorrtTAexpression.Incontrast,consecutivetransfectionshaveseveraladvantages.Mostimportantly,theresponseplasmidgenerallywillnotcointegratewiththeregulator,andyoucanselectadouble-stablecelllinethatgivesverylowtonobackgroundexpressionofGeneX.Furthermore,onceyouhavedevelopedasuitableTet-OfforTet-Oncellline,itprovidesaprovengenetic background into which you can introduce many different responseplasmids.
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II. Protocol Overview continued
Figure 4. Overview of developing Tet-Off and Tet-On and double-stable Tet-Off and Tet-On cell lines. TousetheTetGeneExpressionSystems,youwillneedtomakea"double-stable"Tetcellline,asoutlinedabove.Ifyouarestartingwithyourowncellline,youwillneedtoperformtheentireprocedureoutlinedabove.IfyouarestartingwithoneofourpremadeTet-OfforTet-OnCellLines,onlyperformthesecondstabletransfection.
FIRST STABLE TRANSFECTION(Section VIII; ~ 2 months)
• Transfect with regulator plasmid (pTet-Off or pTet-On)
• Select G418-resistant clones
• Screen by transient transfections with pTRE2hyg-Luc for clones with low background and high Tc- or Dox- dependent induction
SECOND STABLE TRANSFECTION(Section IX; ~ 2 months)
• Transfect with response plasmid; cotransfect with Linear Marker (or pTK-Hyg or pPUR), if necessary
• Select hyg- or puro-resistant clones
• Screen by a gene-specific assay for clones with low background and high Tc- or Dox-dependent induction of Gene X
pTet-Off(or pTet-On)
(r)tTANeor
Double-stable Tet-Off or Tet-On cell line
Tet-Off or Tet-On cell line(Premade cell lines are available
from Clontech)
Host cell line
Hygr/Purr
Perform pilot experiments
(Section VII; ~ 3 weeks)
Regulatoryplasmid
pTREor
pTRE-Tight
Gene X
Responseplasmid
pTRE2hyg/pur
Gene X
+OR
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III. List of Components
Storeallmammaliancelllinesinliquidnitrogen(–196°C).StoreallplasmidsandFetalBovineSerumat–20°C.VisitourTetSystemsproductpagewww.clontech.com foracurrentlistofcelllinesandproductsavailablefortheTetSystems.
Tet-Off® Gene Expression System (Cat. No. 630921)• 20 µl pTet-OffVector(0.5µg/µl)• 20 µl pTRE2hygVector(0.5µg/µl)• 20 µl pTRE2hyg-LucVector(0.5µg/µl)• 0.5 ml CHO-AA8-LucTet-OffControlCellLine(1x106cells)• 50 ml TetSystemApprovedFetalBovineSerum• pTRE2hygVectorInformationPacket(PT3521-5)• TetCellLinesProtocol-at-a-Glance(PT3001-2)
Tet-On® Gene Expression System (Cat. No. 630922)• 20 µl pTet-OnVector(0.5µg/µl)• 20 µl pTRE2hygVector(0.5µg/µl)• 20 µl pTRE2hyg-LucVector(0.5µg/µl)• 0.5 ml U2-OS-LucTet-OnControlCellLine(1x106cells)• 50 ml TetSystemApprovedFetalBovineSerum• pTRE2hygVectorInformationPacket(PT3520-5)• TetCellLinesProtocol-at-a-Glance(PT3001-2)
Tet-Off® Cell Lines• 1.0 ml Tet-OffCellLine(2x106cells/ml)• 0.5 ml CHO-AA8-LucTet-OffControlCellLine(1x106cells)• 50 ml TetSystemApprovedFetalBovineSerum• TetCellLinesProtocol-at-a-Glance(PT3001-2)
Tet-On® Cell Lines• 1.0 ml Tet-OnCellLine(2x106cells/ml)• 0.5 ml U2-OS-LucTet-OnControlCellLine(1x106cells)• 50 ml TetSystemApprovedFetalBovineSerum• TetCellLinesProtocol-at-a-Glance(PT3001-2)
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IV. Additional Materials Required
For cell culture • Dulbecco’s Modified Eagle’s Medium(DMEM,Sigma,Cat.No.D5796),
Alpha Minimal Essential Medium Eagle (alpha-MEM),RPMI-1640,orother specified medium.The appropriate medium for growing ClontechpremadeTet-OffandTet-OncelllinesisdescribedontheProductAnalysisCertificateprovidedwitheachcellline.
• Fetal bovine serum (FBS) It iscritical that theFBSnot inhibitTet-responsiveexpression.Youcan
eliminateTccontaminationproblemsbyusingClontechTetSystemApprovedFBS(seeRelatedProducts).ThisserumhasbeenfunctionallytestedintheTetSystemstoensureagainstpossibleTccontamination.Alternatively,usetheCHO-AA8-LucControlCellLinetotestforTccontaminationinothersera,asdescribedinSectionVII.A.
Note: The PC-12 Tet-Off and Tet-On Cell Lines require horse serum(SigmaCat.No.0146)forgrowth,whichdoesnotnormallycontainTc.
• 200 mM L-Glutamine(Sigma,Cat.No.G7513)
• Solution of 10,000 units/ml Penicillin G sodium and 10,000 µg/ml Streptomycin sulfate (Sigma,Cat.No.P0781)
• Antibiotics for clonal selection Prior to use, determine the optimal concentration of each antibiotic for
selectionasdescribedinSectionVII.B. G418 (forselectionofTet-OffandTet-OnCellLines) G418isavailableinpowderedformfromClontech(Cat.No.631307).Note
thattheeffectiveweightisabout0.7gpergramofpowder.Makea10mg/mlstocksolutionbydissolving1gofpowderinapproximately70mlofDMEMoralpha-MEM(withoutsupplements).Filtersterilizeandstoreat4°C.
Recommended working concentration: Maintenance:100µg/ml Selection(HeLaorCHOcells):400–500µg/ml
(acceptablerange):50–800µg/ml Hygromycin (forselectionofdouble-stableTet-OffandTet-OnCellLines)
HygromycinBisavailablefromClontech(Cat.No.631309). Recommended working concentration: Maintenance:100µg/ml Selection(HeLaorCHOcells):200µg/ml
(acceptablerange):50–800µg/ml Puromycin (for maintenance of the MDCK Tet-Off Cell Line and for
selectionofdouble-stableTet-OnandTet-Offcells)availablefromClontech(Cat.No.631305&631306)
Recommended working concentration: Maintenance:0.5µg/ml Selection(acceptablerange):0.5–5µg/ml
• Trypsin-EDTA(Trypsin;Sigma,Cat.No.T3924)
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IV. Additional Materials Required continued
• Dulbecco’s phosphate buffered saline(DPBS;Sigma,Cat.No.D8662)
• Cell Freezing Medium, withorwithoutDMSO(Sigma,Cat.No.C6164orCat.No.C6039)
• Tissue culture plates and flasks, availablefromBDDiscoveryLabware(www.bdbiosciences.com/discovery_labware)
• Cloning cylinders or discs(PGCScientific,Cat.No.62-6150-40,-45orCat.No.62-6151-12,-16)
For transient and stable transfections
Thetransientandstabletransfectionsinthisprotocolcanbeperformedbyvariousmethods.Reagentswilldependonwhichtransfectionmethodyouuse.AlthoughwegenerallyuseelectroporationforbothtransientandstabletransfectionswiththeTet-OffandTet-OnSystem,othermethodsworkwellandmaybepreferable,dependingoncelltype.
The CalPhos™ Mammalian Transfection Kit (Cat. No. 631312) andClonfectin™ Transfection Reagent (Cat. No. 631301), are available forhigh-efficiencycalcium-phosphateorliposome-mediatedtransfections.
The efficiency of transfection for different cell lines may vary greatly.Amethodthatworkswellforonehostcelllinemaybeinferiorforanother.Therefore,whenworkingwithacelllineforthefirsttime,youmaywanttocomparetheefficienciesofseveraltransfectionprotocols.Youcantransfectthehostcelllinewithanoninduciblereporter/expressionvector,suchaspCMVβ(Cat.No.631719)orpAcGFP1-N1(Cat.Nos.632469&632426)andassayforreportergeneactivity.
Afteramethodoftransfectionischosen,itmaybenecessarytooptimizeparameterssuchascelldensity,theamountandpurityoftheDNA,mediaconditions,andtransfectiontime.Onceoptimized,theseparametersshouldbekeptconstanttoobtainreproducibleresults.
If cotransfection is required to create a stable cell line with your pTREvector, we recommend cotransfection with Linear Hygromycin Marker(Cat.No.631625)orLinearPuromycinMarker(Cat.No.631626).Thesemarkersareshort,purifiedlinearDNAfragmentscomprisedofthemarkergene,anSV40promoter,andtheSV40polyadenylationsignal.Becauseoftheirsmallsize,thesemarkersarehighlyeffectiveatgeneratingstabletransfectants.Alternatively,youcanusepTK-HygVector(Cat.No.631750)orpPURVector(Cat.No.631601).
Note: IfyouareusingaselectionvectorotherthanaLinearSelectionMarker,pTK-Hyg,orpPUR,thepromotershouldnotcontainanenhancerelement.Ifitdoes,cointegrationoftheresponseandselectionplasmidsmayleadtohighbackgroundexpressionofGeneXintheuninducedstate.
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For regulation of gene expression
• Doxycycline (Cat.No.631311).Diluteto1–2mg/mlinH2O.Filtersterilize,aliquot,andstoreat–20°Cinthedark.Usewithinoneyear.
• Tetracycline hydrochloride(SigmaCat.No.T3383)Diluteto1mg/mlin70%ethanol.Filtersterilize,aliquot,andstoreat–20°Cinthedark.Usewithintwomonths.
For luciferase assays• Useanystandardluciferaseassaysystem.WerecommendourLuciferase
ReporterAssayKit(Cat.No.631714).
For PCR confirmation of integrated plasmids (optional)
• If you wish to confirm the presence of integrated plasmids in clonalhygromycin-,puromycin-,orneomycin-resistantcelllines,youwillneedtodesignPCRprimersthatamplifyaportionoftheappropriateregulatororresponseplasmid.
IV. Additional Materials Required continued
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V. Plasmid Manipulations
A. Propagation of Vector Plasmids 1.TransformeachoftheplasmidsprovidedinthiskitintoasuitableE. coli
hoststrain(e.g.,DH5α)toensurethatyouhavearenewablesourceof DNA.Tet vectors are low copy-number, so use chloramphenicolamplificationtoincreaseplasmidyields.
2.You will need to perform large-scale plasmid preparations of anyplasmid thatwillbe introduced intomammaliancells.Toensure thepurityoftheDNA,preparetransfection-gradeplasmidbypurificationon a NucleoBond® column. Visit www.clontech.com for completeproductinformation.
B. Generating Your Gene-Specific Expression Vector GenerateyourpTRE-GeneXconstructusingstandardmolecularbiology
techniques,asdescribedbelow.Formoredetailedinformation,seeSambrooket al. (2001).
1.Purify the Gene X fragment by any standard method, such as theNucleoTrapGelExtractionKit(Cat.No.636018)orNucleoTrapPCRPurificationKit(Cat.No.636020).ThecDNAorgenefragmentmustcontainanATGinitiationcodon.Insomecases,additionofaKozakconsensusribosomebindingsite(Kozak,1987)mayimproveexpressionlevels;however,manygeneshavebeenefficientlyexpressedinTetsystemswithouttheadditionofaKozaksequence.Thefragmentcanbegeneratedusingcompatiblerestrictionsitesthatarepresentoneithersideofthegeneandinthecloningvector.Ifnosuchsitesarepresent,thegenefragmentcanbegeneratedbyPCRwithsuitablerestrictionsitesincorporatedintotheprimers.
2.Digesttheresponsevector(pTREoritsvariant)withtheappropriaterestrictionenzyme(s),treatwithphosphatase,andpurify.
3.LigatetheresponsevectorandtheGeneXfragment. 4.TransformligationmixturesintoE. coli. 5.Identifythedesiredrecombinantplasmidbyrestrictionanalysis,and
confirmorientationandjunctionsbysequencing.
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VI. Cell Culture Guidelines
A. General Information The protocols in this User Manual provide only general guidelines for
mammaliancellculturetechniques.Performallstepsinvolvingcellcultureusing sterile technique in a suitable hood. For those requiring moreinformationonmammaliancellculture,werecommendthefollowinggeneralreferences:
• Culture of Animal Cells,FourthEdition,ed.byR.I.Freshney(2000,Wiley-Liss,NY)
• Current Protocols in Molecular Biology, ed. by F. M.Ausubel et al. (1995,Wiley&Sons)
B. Characteristics of Tet-Off and Tet-On Cell Lines See the Product Analysis Certificate (PAC) for information on each
Tet-Off andTet-On Cell Line.Additional information for all the currentlyavailableTet-OffandTet-OnCellLines,includingpropagationinformation,is provided in documents PT3001-2 and PT3001-3, available from ourTetSystemsproductpageatwww.clontech.com.
General cell culture conditions:PremadeTet-OffandTet-OnCellLinesshouldbegrownat37°Cinahumidifiedchamberwith5–10%CO2.SeethePACfordetailsparticulartoeachcellline.
Relative growth rates:TheincubationtimesinthisUserManualareforcellssuchasCHOorHeLawithrelativelyrapiddoublingtimes.Othercelltypeswilldifferintheirgrowthrates.
Selection in G418 and hygromycin:Maintainstableanddouble-stableTet-OffandTet-OnCellLinesintheappropriateselectivemedium;however,theconcentrationcanbereduced(typically to100µg/ml foreachdrug)fromthelevelsusedtoselectstablytransfectedclones.Youmaywishtoalternatebetweenselectingandnonselectingconditions.
C. Starting Tet Cell Cultures From Frozen Stocks Note: Frozencellsshouldbeculturedimmediatelyuponreceiptorassoon
thereafteraspossible.Increasedlossofviabilitymayoccuraftershippingifculturingisdelayed.
1.Thawvialofcellsrapidlyina37°Cwaterbathwithconstantagitation.Immediatelyuponthawing,wipetheoutsideofthevialwith70%EtOH.Transferthecontentsofthevialtoa10-cmdish,oraT25orT75flask,containing1mlofmedium(withoutantibiotics).Mixgently.
2.Addanadditional4mlofmediumtotheflask/dishandmixgently. 3.Addadditionalmediumtothecultureasfollows: T25flaskor10-cmdish add5ml T75flask add10ml
Note:ForJurkatandothersuspensioncultures,suspendcellsatadensityofnolessthan2x105cells/mlintheappropriatemedium.
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VI. Cell Culture Guidelines continued
4.Mixthecellsuspensionthoroughly.Gentlyrockorswirlthedish/flasktodistributethecellsevenlyoverthegrowthsurfaceandplaceitina37°Chumidifiedincubator(5–10%CO2asappropriate).
5.[Alternativemethod]Thecellscanalsoberinsedpriortoincubation.Ifrinsingisdesired,performsteps1and2ina15-mlconicalcentrifugetube.Centrifugeat125xg for10min,and resuspend incompletemediumforculturing.Thisstepremovesthecryopreservativeandcanbebeneficialwhenresuspendinginsmallvolumes.However,thisstepcandamagefragilecellmembranes.
6.Thenextday,examinethecellsunderamicroscope.Ifthecellswerenotrinseduponthawing(step5),centrifugecells(ifsuspensioncultures),aspirate themedium,and replacewith fresh,prewarmed, completemedium(withoutantibiotics).
7.Expand the culture as needed. Note: The appropriate selectiveantibiotic(s)maybeaddedtothemediumafter48–72hrinculture.
D. Preparing Frozen Stocks of Tet Cell Lines Once you have started growing a Tet-Off or Tet-On Cell Line from
Clontech, prepare frozen aliquots to ensure a renewable sourceof cells. Similarly, prepare frozen aliquots of any double-stableTet-Off orTet-On cell line or of anyTet-Off orTet-On cell line that youmake.
1.Trypsinizethedesirednumberofflasks. 2.Poolcellsuspensionstogether,countcells,andcalculatetotalviable
cellnumber. 3.Centrifugecellsat125xgfor10min.Aspiratethesupernatant. 4. Resuspend the pellet at a density of at least 1–2 x106 cells/ml
in freezing medium. Freezing medium can be purchased from Sigma(Cat. No. C6164), or freeze cells in 70–90% FBS, 0–20% medium(noadditives),and10%DMSO.
5.Dispense1-mlaliquotsintosterilecryovials. 6.Freeze slowly (1°C per min). Nalgene makes cryo-containers
(NalgeneCat.No.5100) for thispurpose ifaspecialized freezer isnot available (freeze at –80°C overnight). Alternatively, place vialsin a thick-walled styrofoam container at –20°C for 1–2 hr.Transferto –80°C overnight. Remove vials from styrofoam container orcryo-containersthefollowingdayandplaceinliquidnitrogenstorageorultralow-temperaturefreezer(–150°C).
7.(Twoormoreweekslater)Plateavialoffrozencells,asdescribedinSectionC,toconfirmviability.
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VII. Pilot Experiments
5 x 103
10 x 103
15 x 103
Fold
-ind
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Tet SystemApproved FBS
Other commerciallyavailable FBS
Figure 5. Fold induction of luciferase activity in different lots of FBS. TheCHO-AA8-LucTet-OffControlCellLinewasgrowninmediapreparedwithdifferentlotsofFBS.Averageuninducedexpressionlevel=0.21RLU(n=21,S.D.=0.07);maximumexpressionlevelsvariedfrom123to3,176RLU.
A. Pilot Experiment with the CHO-AA8-Luc Tet-Off or U2-OS-Luc Tet-On Control Cell Line
Beforeyouperformanyotherexperiments,westronglyrecommendthatyouperformadose-responsecurvewiththeCHO-AA8-LucTet-OffControlCell Line provided with Tet-Off kits or the U2-OS-Luc Tet-On Cell LineprovidedwiththeTet-Onkits.Thesepremadedouble-stableTetCellLinescanexhibitover104-foldinductionofluciferaseuponremoval(Tet-Off)oraddition(Tet-On)ofTcorDoxfromtheculturemedium(Figure5).Inadditiontoprovidinga"hands-on"introductiontotheTetSystems,thisexperimentservestwocriticalfunctions:
• Determination of effective concentrations of Tc or Dox stocks:TheconcentrationsofTcandDox listed throughout thisprotocolareapproximate.Theoptimalconcentrationmayvarywithdifferentcelllinesandwithdifferent lotsofantibiotic. Ingeneral, full repressionofgeneexpressioninTet-Offcelllinescanbeobtainedwith1–2µg/mlTcor10ng–1µg/mlDox.FullactivationofgeneexpressioninTet-Oncelllinescanbeobtainedwith100ng–1µg/mlDox.
• Testing of serum for Tc contamination:AsshowninFigure5,differentlotsofFBSexhibitsignificantvariation in theireffectonTetSystemexpression,presumablyduetothewidespreaduseoftetracyclinesinthedietofcattle.The~10,000-foldinductionofluciferaseinCHO-AA8-LucTet-OffControlCellsinresponsetoTcorDoxishighlyreproducible.Ifyouseeasignificantlylowerlevelofinduction(e.g.,100–1,000-foldorless),thismaysuggestthatyourserumcontainsTc.Thistestshouldberepeatedwitheachdifferentlotofserum.Alternatively,useTetSystemApprovedFBS(Cat.No.631101orCat.No.631106),whichhasbeenfunctionallytestedandshowntoallowthefullrangeofinductionpossiblewiththeTetSystemcelllines.
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VII. Pilot Experiments continued
Procedure: 1.Plate6aliquotsof0.5x105CHO-AA8-LucTet-OfforU2-OS-LucTet-On
cellseachinto5mlofcompleteculturemediumin6-wellculturedishes. 2.To titrateTc:addTc to final concentrationsof0, 1 x10-4, 1 x10-3,
1x10-2,0.1,1.0,and10.0µg/ml. TotitrateDox,addDoxtofinalconcentrationsof0,1x10-3,1x10-2,
0.1,1.0,10,and100ng/ml. 3.Allowthecellstogrowfor48hr. 4.Assayeachsampleforluciferaseactivityusinganystandardluciferase
assay.PlotyourresultslogarithmicallyandcomparetoFigure6. B. Titrating G418, Hygromycin, and Puromycin (Kill Curves) Prior to using G418, hygromycin, or puromycin to establish stable and
double-stablecelllines,itisimportanttotitrateyourselectionagentstockstodeterminetheoptimalconcentrationforselectionwiththeparticularhostcelllinebeingtested.Thisisalsoimportantbecauseoflot-to-lotvariationinthepotencyofthesedrugs.Therefore,youshouldtitrateeachnewlotofantibiotictodeterminetheoptimalconcentration.Werecommendthatyouperformtwoexperimentsforeachdrug:(1)atitrationtodeterminetheoptimaldrugconcentration,and(2)anexperimenttodeterminetheoptimalplatingdensity.ThisstepisrecommendedevenifyouareusingpremadeTetCellLines.
1.Titrateatfixedcelldensity. a.Plate 2 x 105 cells in each of six 10-cm tissue culture dishes
containing10mloftheappropriatecompletemediumplusvaryingamounts(0,50,100,200,400,800µg/ml)ofhygromycinorG418.Forpuromycin,addthedrugat0,1,2.5,5,7.5,and10µg/ml.
Note:293Tet-OnandTet-Offcells(Cat.No.630903andCat.No.630908,respecitively)areespeciallysensitivetohygromycin;testaconcentrationrangewithamidpointof25µg/ml.Saos-2Tet-Offcells(Cat.No.630911)exhibitresistancetohygromycin;testaconcentrationrangewithamidpointof800µg/ml.
b. Incubatethecellsfor10–14days,replacingtheselectivemediumeveryfourdays(ormoreoftenifnecessary).
c. Examinethedishesforviablecellseverytwodays.
Forselectingstabletransformants,usethelowestconcentrationthatbeginstogivemassivecelldeathin~5daysandkillsallthecellswithintwoweeks.ForHeLaandCHOcells,wehavefound400µg/mlG418and200µg/mlhygromycintobeoptimal.Inmammaliancellstheoptimallevelofpuromycinistypicallyaround1µg/ml.
2.Determineoptimalplatingdensity.
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VII. Pilot Experiments continued
3.Onceyouhavedeterminedtheoptimaldrugconcentration,determinetheoptimalplatingdensitybyplatingcellsatseveraldifferentdensitiesinthepresenceofaconstantamountofdrug.Ifcellsareplatedattoohighadensity,theywillreachconfluencybeforetheselectiontakeseffect.Optimalplatingdensityisdependentonpopulationdoublingtimeandcellsurfacearea.Forexample,largecellsthatdoublerapidlyhavealoweroptimalplatingdensitythansmallcellsthatdoubleslowly.
a.Platecellsatseveraldifferentdensitiesineachofsix10-cmtissueculturedishescontaining10mloftheappropriateselectivemedium.Suggesteddensities(cells/10-cmdish):5x106,1x106,5x105,2x105,1x105,and5x104.
b. Incubatethecellsfor5–14days,replacingtheselectivemediumeveryfourdays.
c. Examinethedishesforviablecellseverytwodays. Forselectingstabletransfectants,useaplatingdensitythatallowsthe
cellstoreach~80%confluencybeforemassivecelldeathbegins(ataboutday5).Thisisthecelldensityatwhichcellsshouldbeplatedforselectionofstable transfectants.ForHeLacells,wehave found2x105cells/10-cmdishtobeagoodplatingdensity.
Figure 6. Dose-response curves for the Tet Control Cell Lines. Panel A. Dose-responsecurves for the CHO-AA8-Luc Control Cell Line. Experiments with another control cell line(CHO-K1-EGFP-Luc Tet-Off) have demonstrated that suppression can be maintained with Doxconcentrationsaslowas10pg/ml(Cunninghamet al. ,1997).Differencesinbackgroundandinductionlevelscanoccurbetweenmultipleindependentclonallineswhenestablishingdouble-stableTet-OffCellLines(seeSectionIX.C). Panel B.Dose-responsecurvefortheU2-OS-LucTet-OnControlCellLine.
U�-OS-Luc Tet-On Control Cells
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C. [optional] Test Potential Host Cells by Transient Transfection with pTRE2hyg-Luc and pTet-Off or pTet-On
Tet expression systems have been established in numerous cell linesincludingHeLa,CHO,MCF7,HEK293andHepG2.However,thesystemmaynotbecompatiblewitheverycelltype.PerformingatransientexpressionassaywithpTet-Off(orpTet-On)andpTRE2hyg-LucmayprovideaquickindicationofwhetherornottheTetsystemswillworkinaparticularcellline.ThistestisnotnecessaryifyouhavepurchasedapremadeTet-OfforTet-OnCellLine.
YoushouldtransfectcellsusingvaryingratiosofpTet-Off/OntopTRE2hyg-Luc.Forexample,try:
pTet-Off/On : pTRE2hyg-Luc 1µg : 1µg 1µg : 10µg 10µg : 1µg
Important Note:Fold-inductionlevelsarealmostalwayslowerintransientassaysthaninproperlyscreenedstableanddouble-stablecelllines.Forexample,theSaos-2Tet-OffCellLineexhibits~40-foldinductionintransientexpressionassays,butstableclonescanbeisolatedthatexhibit6,000-foldinductionandbackgroundexpressionlevelsthatareindistinguishablefromcontrolbackgroundexpression.Therefore,anapparentlackofinductionresponseinthetransientassayshouldnotbethesolereasonforabortingyourexperimentsinaparticularcellline.
VII. Pilot Experiments continued
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VIII. Development of Stable Tet Cell Lines
Skip Section viii if you have purchaSed a premade tet-off or tet-on cell line
A. Transfection and Selection of Stable Cell Lines (Figure7) The following protocol describes the development of Tet-Off or Tet-On
celllines.Youmustoptimizetheprotocolforeachcelltype.Someoftheparametersmostlikelytoneedadjustmentare:platingdensities,transfectionmethod,G418concentrations forselection,and incubationandgrowingtimes.
Regardlessofthecelltypeandtransfectionmethod,thegoalistogenerateacelllinethatgiveslowbackgroundandhighinductionofluciferaseactivitywhen testedby transient transfectionwithpTRE2hyg-Luc inSectionB.BecausethelevelofexpressionoftTAorrtTAisprofoundlyaffectedbythesiteofintegration,werecommendthatyouisolateandanalyzeasmanyclonesaspossibleatStep6.Ingeneral,testatleast30clones.Wehavescreenedasmanyas100clonestoobtainonethatexhibitssuitablyhighinductionandlowbackground.
1.Growcells to~80%confluency incompletemediumor toadensityappropriateforyourtransfectionmethod.
2.TransfectthepTet-OnorpTet-OffVectorbythedesiredmethod.Note:Ifdesired,theregulatorplasmidcanbelinearizedbydigestionwitharestrictionenzyme(ScaIforpTet-On/Off).
3.Plate transfectedcells in ten10-cmculturedishes,eachcontaining10 ml of the appropriate complete medium, at the optimal densitydeterminedinSectionVII.
4.Allowcellstodividetwice(24–48hr),thenaddG418to400–500µg/ml.Note:TheexactconcentrationofG418forselectionandtheoptimalplatingdensitymayvaryfromcelltypetocelltypeandwithdifferentlotsofG418.SeeSectionVII.B.
5.ReplacemediumwithfreshcompletemediumplusG418everyfourdays,ormoreoftenifnecessary.
Afteraboutfivedays,cellsthathavenottakenuptheplasmidshouldstarttodie.Splitthecellsiftheyreachconfluencybeforemassivecelldeathbegins.
After2–4weeks,isolatedcoloniesshouldbegintoappear. 6.Isolatelarge,healthycoloniesandtransferthemtoindividualplatesor
wells.Suspensionculturesmustbeclonedusingthelimitingdilutiontechnique.WhenworkingwithadherentcellsatClontech,wegenerallyisolateclonesusingcloningcylindersorcloningdiscs.
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Figure 7. Flow chart for developing Tet cell lines.
• Transfect host cell line with regulator plasmid (pTet-Off or pTet-On)
• Select in presence of G�1�
• Isolate at least 30 G�1�- resistant clones
• Screen by transient transfections with pTRE�hyg-Luc for clones with low background and high induction of luciferase in response to Tc or Dox
• Freeze stocks of Tet cell line
Hostcell line
Tet-Off or Tet-Oncell line
pTet-OffOR
tTA Neor
pTet-On
rtTANeor
VIII. Development of Cell Lines continued
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VIII. Development of Cell Lines continued
B. Screening Stable Cell Lines ThenextstepistoperformtransienttransfectionassayswithpTRE2hyg-Luc
(oranotherreportervector)toidentifyG418-resistantclonesthatmeetthecriteriaforstableTet-OfforTet-Oncelllines.SeeAppendixAformapsandmoreinformationonthesereportervectors.
1.Pick clones and expand as needed for your particular cell line.Screenclonesoncetheyreach50–80%confluencyina6-wellplate.
2.Trypsinizethecellsandsplitabout1/3 intoasinglewellofa6-wellplate.Thecellsinthis"stockplate"willbepropagateddependingupontheresultsofthescreeningassay.
3.Transfecttheremaining2/3ofthecellswith1–2µgofpTRE2hyg-Lucor another reporter vector, using the desired transfection method.Decrease the amount of DNA if performing liposome-mediatedtransfection.Splitintotwowellsofasix-wellplate.
4.AddDox(1–2µg/ml)tooneofthetwowellsfromstep3. 5.Incubatethetransfectedcellsfor48hr. 6.Assayforinduction: LuciferaseAssay:calculatefold-induction ForTet-Off:Fold-induction=–DoxRLU/+DoxRLU ForTet-On:Fold-induction=+DoxRLU/–DoxRLU 7.Selectcloneswiththehighestfold-induction(highestexpressionwith
lowestbackground)forpropagationandfurthertesting.Ingeneral,onlyselectclonesthatexhibit>20-foldinduction.
8.Freezestocksofeachcloneassoonaspossibleafterexpandingtheculture.
Note:SomeresearchersmaydesiretoconfirmthepresenceofthetTAandrtTAregulatoryproteinsinstableTetcelllinesbyWesternanalysiswith theVP16PolyclonalAntibody(Cat.No.631209).Useof theseantibodiesonlyverifiesthepresenceoftTAorrtTA;itdoesnotrevealthefunctionalinducibilityofthesecelllines.Furthermore,tTAandrtTAexpressioninstablecelllinesmaybebelowlevelsdetectablebyWesternblotting.HighlevelsoftTAorrtTAarenotrequiredforgoodinduction,and in fact, overexpression of tTA can be toxic to cells.Therefore,WesternanalysisshouldNOTsubstituteforthefunctionalscreen.
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IX. Development of Double-Stable Tet Cell Lines
A. Test pTRE-Gene X by Transient Transfection into a Tet-Off or Tet-On Cell Line
Prior toestablishingyourdouble-stableTet-OfforTet-Oncell lines,youshouldtestyourpTRE-GeneX(orpBITetVector)constructforfunctionality.TransientlytransfectpTRE-GeneXintothecelllinecreatedinSectionVIII,orthepremadeClontechTetCellLine.IfyouarenotusingapBIVectororoneofthetaggedvectors(pTRE-Myc,-HAor-6xHN),youwillneedtodesignagene-specificassaytotestfortheinductionofGeneX.Examplesofgene-specificassaysthatcanbeusedinclude:
• WesternblotwithanantibodytoProteinX • RT-PCRusingGeneXprimers.BesureyoucandiscriminatePCR
productsgeneratedfromgenomicDNAfromtrueRT-PCRproducts. • NorthernblotwithGeneXprobe • FunctionalassayforProteinX
B. Stably Transfect and Select Double-Stable Cell Lines (Figure8) Thenextstep is tostably transfect thestable(orpremade)Tetcell line
withyourpTRE-GeneXconstruct.Thegoalistogenerateacelllinethatgives low background and high expression of Gene X when tested inSection IX.D. Both expression levels and induction of Gene X can beprofoundlyaffectedbythesiteofintegration.Insertionnearanenhancermayresult inhighbasalexpressionofGeneX,whereasother insertionsitesmayresultinsuboptimalinduction.Tofindtheclonewiththehighestinductionandlowestbackground,werecommendthatyougrowandanalyzeasmanyclonesaspossible.Ingeneral,testatleast30clones.Wehavescreenedasmanyas100clonestoobtainonethatexhibitssuitablyhighinductionandlowbackground.
IMPORTANT:IfyouarenotusingpTRE2hyg,pTRE2puroranotherresponsevectorbearingamammalianselectionmarker,skipthestepsbelowandusethecotransfectionprotocolinSectionIX.C.
1.Growcells to~80%confluency incompletemediumor toadensityappropriateforyourtransfectionmethod.
2.TransfectcellswithpTRE2hyg-GeneXorpTRE2pur-GeneX.Note:Ifdesired,theplasmidscanbelinearizedbydigestionwitharestrictionenzyme(checktheVectorInformationPacketsprovidedwitheachvectorforappropriaterestrictionsites).
3.Plate transfectedcells in ten10-cmculturedishes,eachcontaining10 ml of the appropriate complete medium, at the optimal densitydeterminedinSectionVII.
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Tet Systems User Manual
IX. Development of Double-Stable Tet Cell Lines continued
Figure 8. Flow chart for developing double-stable Tet cell lines.
• Insert Gene X into pTRE2hyg/pur, another pTRE variant, or a pBI vector
• Transfect Tet-Off or Tet-On cell line with pTRE2hyg/pur-Gene X response plasmid; or cotransfect pTRE-Gene X or pBI-Gene X with a Linear Marker
• Select in presence of hygromycin or puromycin (Tc or Dox should be included in the medium when establishing double-stable Tet-Off cell lines.)
• Isolate at least 30 hygromycin/puromycin-resistant clones
• (OPTIONAL) Confirm presence of integrated pTRE-Gene X in clones by PCR
• Screen by a gene-specific assay for clones with: – Low background of Gene X – High induction of Gene X
Possible assays: – Western blot using an antibody to Protein X – RT-PCR using Gene X primers – Northern blot with Gene X probe – Functional assay for Protein X – Reporter activity (β-galactosidase, or luciferase on pBI vector)
• Freeze stocks of double-stable cell lines
Double-stableTet-Off or Tet-On
cell line
pTRE2hyg/pur
Gene X
Tet-Off or Tet-Oncell line
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IX. Development of Double-Stable Cell Lines continued
4.Allow cells to divide twice (24–48 hr), then add the appropriateselectionagent,hygromycinorpuromycin,totheoptimalconcentrationdetermined in Section VII. For hygromycin the range is generally200–400µg/mlandforpuromycinitis1–5µg/ml.
For Tet-Off cells only: When establishing a double-stable Tet-Offcellline,werecommendthatyouculturethecellsinthepresenceof2µg/mlTcor1µg/mlDoxinordertokeeptranscriptionofGeneXturned“off”.ThisisessentialifProteinXistoxictothecell.
5.Replacemediumwithfreshcompletemediumcontainingtheselectionantibiotic(hygorpur)everyfourdays.FreshDoxMUSTbeaddedeverytwodaysforTet-Offcells.
Afteraboutfivedays,cellsshouldstarttodie.Splitcellsiftheyreachconfluencybeforemassivecelldeathbegins.
After2–4weeks,hyg-resistantorpur-resistantcolonieswillbegintoappear.
6.Isolatelarge,healthycoloniesandtransferthemtoindividualplatesorwells.Isolateasmanyclonesaspossible.
7.ProceedtoSectionIX.D.
C. Stably Transfect and Select Double-Stable Cell Lines—Cotransfection pTRE2hyg-GeneXandpTRE2pur-GeneXresponseplasmidscontaina
selectionmarkerinthebackbone.OtherpTREresponseplasmidswhichdo not contain a marker must be cotransfected with a selection vectorsuchasaLinearSelectionMarker,pTK-Hyg,orpPURusingthefollowingprotocol.
Note: IfyouareusingaselectionvectorotherthanaLinearSelectionMarker,pTK-Hyg,orpPUR,thepromotershouldnotcontainanenhancerelement.Ifitdoes,cointegrationoftheresponseandselectionplasmidsmayleadtohighbackgroundexpressionofGeneXintheuninducedstate.
1.Growcells to~80%confluency incompletemediumor toadensityappropriateforyourtransfectionmethod.
2.TransfectpTRE-GeneXandaLinearSelectionMarker,pTK-Hyg,orpPURinaratioofbetween10:1and20:1bythedesiredmethod.Youmaywanttooptimizeratios.Note:Ifdesired,theplasmidscanbelinearizedbydigestionwitharestrictionenzyme(checktheVectorInformationPacketsprovidedwitheachvectorforappropriaterestrictionsites).
3.Platetransfectedcellsinten10-cmculturedishes,eachcontaining10 ml of the appropriate complete medium, at the optimal densitydeterminedinSectionVII.
4.Allow cells to divide twice (24–48 hr; time may vary with cell line),thenaddhygromycin(orpuromycin)to200–400µg/ml(ortheoptimalconcentrationdeterminedinSectionVII).
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IX. Development of Double-Stable Cell Lines continued
For Tet-Off cells only: Whenestablishingadouble-stableTet-Offcellline,youmaywishtoculturethecellsinthepresenceof2µg/mlTcor1µg/mlDoxinordertokeeptranscriptionofGeneXturned“off”.ThisisessentialifProteinXistoxictothecell.
5.Replacemediumwithfreshcompletemediumcontaininghygromycin(orpuromycin)everyfourdays.FreshDoxMUSTbeaddedeverytwodaysforTet-Offcells.
Afteraboutfivedays,cellsshouldstarttodie.Splitcellsiftheyreachconfluencybeforemassivecelldeathbegins.
After2–4weeks,hyg-(orpuro-)resistantcolonieswillbegintoappear. 6.Usingcloningcylindersordiscs, isolate large,healthycoloniesand
transferthemtoindividualplatesorwells.Isolateasmanyclonesaspossible.
D. Screening Double-Stable Cell Lines 1.Test isolated resistant clones for Dox-regulated gene expression
bydividingasuitablenumberofcellsinhalfandtestingforGeneXexpression(orpBIreporterexpression)inthepresenceandabsenceof1µg/mlDox.
Aswith thedevelopmentofTet-OfforTet-Oncell lines, youshouldgenerallychoosethecelllinethatgivesyouthehighestoverallinductionandlowestbackground(i.e.,uninducedexpressionlevel)ofGeneX.
2.Allowthecellstogrowforatleast48hr,thenassayeachsampleforGeneXexpressionusingoneofthemethodsdescribedinSectionA.
3.[Optional]ConfirmthepresenceofintegratedpTRE-GeneXbyperformingPCRonchromosomalDNAusingprimersthatwillamplifyaninternalportionoftheplasmid.
4.Onceyouhavedevelopedasuitabledouble-stableTet-OfforTet-Oncellline,preparefrozenaliquotstoensurearenewablesourceofthecells(SectionVI.D).
E. Working with Double-Stable Tet Cell Lines TheTetSystemhasbeenestablishedsuccessfullyinmanycelltypes,as
wellastransgenicmice,rats,plants,andyeast.Ingeneral,failuretoobtainacell linewitha lowbackgroundlevelofGeneXexpressionisaresultoftheintegrationsiteinthetestedlines,andcanbeovercomesimplybyscreeningmoreclones.
Performadose-responsecurvesimilar to theexperimentsdescribed inSectionVII.A.ThekineticsofinductionaredependentonthestabilityofthemRNAandprotein.Itmaytakesometimebeforestablyexpressedproteinsaccumulatetoequilibriumlevels.RefertotheresultsseeninFigures1B,3,and6.
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IX. Development of Double-Stable Cell Lines continued
Loss of regulation:Onoccasion,well-characterizeddouble-stablecelllinescanlosetheirresponsivenesstoTcorDox.ThiscanoccurafterchanginglotsofcalforfetalbovineserumandappearstobeduetocontaminationofsomelotsofserumwithTc.Ifyouobserveasuddenlossofresponsiveness,checkyourserumbyperformingadose-responsecurveasdescribedinSectionVII.A.Youcanalsotryreplatingandwashingthecells3hrlatertoremoveanyresidualantibioticthatmaybeinterferingwithinductioncontrol(Rennel&Gerwins,2002).Lossofregulationcanalsobeduetoswitchingofformethylationoftheviralpromoter.Itisrecommendedthatyousubcloneandfreezestocksofyourcellsatvariousstages.
Toxicity of the VP16 activation domain: Some researchers haveinquired about the possible toxic effects of expressing the VP16AD inmammalian cells. In our experience and that of the Bujard laboratoryandthemanyotherlabsthathavesuccessfullyusedtheTetsystem,thishasnotbeenaproblemintissueculture.Likeothertranscriptionfactors,the tTAregulatordoesnothave tobeexpressedathigh levels inordertogiveveryhigh-levelexpressionof thegenes it regulates (i.e., genesencodedontheresponseplasmid).Forexample,GossenandBujardhavecharacterizedHeLaTet-Offcelllinesthatcontain6,000–10,000moleculesoftTApercellandgive105-foldinductionoftheTet-regulatedgenes(pers.comm.).Forin vivoapplications,however,itmaybepreferabletousetheVP16MinimalDomainVectors,whicharetoleratedathigherintracellularconcentrationsandallowactivationoverdifferentranges.SeeAppendixAformoreinformation.
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X. References
YoucanaccessanextensiveTetSystembibliography from theTetSystemsproductpageatwww.clontech.com.Clontech’sTetSystemsweredevelopedincooperationwithDr.BujardandhiscolleaguesattheCenterforMolecularBiologyinHeidelberg(ZMBH).AdditionalbackgroundinformationonTet-regulatedgeneexpressionsystemsisavailableatthesitemaintainedbyDr.Bujard'slaboratory: http://www.zmbh.uni-heidelberg.de/bujard/homepage.htmlPlease note that Clontech is not responsible for the information on, or themaintenanceof,thissite.Ausubel,F.M.,Brent,R.,Kingdom,R.E.,Moore,D.M.,Seidman,J.G.,Smith,J.A.&Struhl,K.,eds.(1995)CurrentProtocolsinMolecular Biology(JohnWiley&Sons,NY).
Baron,U.,Freundlieb,S.,Gossen,M.&Bujard,H.(1995)Co-regulationoftwogeneactivitiesbytetracyclineviaabidirectionalpromoter.Nucleic Acids Res.23:3605–3606.
Baron,U.,Gossen,M.&Bujard,H.(1997)Tetracyclinecontrolledtranscriptionineukaryotes:noveltransactivatorswithgradedtransactivationpotentials.Nucleic Acids Res.25:2723–2729.
Cunningham,S.M.,Cunningham,M.D.,Zhu,L.&Kain,S.(1997)DeterminationandcorrelationofexpressionlevelsofluciferaseandEGFPusingthetetracycline-controlledgeneexpressionsystemandfluorescenceimaging.Neuroscience Abs.23:647.
Freshney,R.I.(2000)Culture of Animal Cells,FourthEdition(Wiley-Liss,NY).
Freundlieb,S.,Schirra-Müller,C.&Bujard,H.(1999)Atetracyclinecontrolledactivation/repressionsystemwithincreasedpotentialforgenetransferintomammaliancells.J. Gene Med.1:4–12.
Gossen,M.,Bonin,A.&Bujard,H. (1993)Control of geneactivity inhighereukaryotic cellsbyprokaryoticregulatoryelements.Trends Biochem. Sci.18:471–475.
Gossen,M.,Bonin,A.L.,Freundlieb,S.&Bujard,H.(1994)Induciblegeneexpressionsystemsforhighereukaryoticcells.Curr. Opin. Biotechnol.5:516–520.
Gossen,M.&Bujard,H.(1992)Tightcontrolofgeneexpressioninmammaliancellsbytetracyclineresponsivepromoters.Proc. Natl. Acad. Sci. USA89:5547–5551.
Gossen,M.&Bujard,H.(1993)Anhydrotetracycline:anoveleffectorfortetracyclinecontrolledgeneexpressionsystemsinhighereukaryoticcells.Nucleic Acids Res.21:4411–4412.
Gossen,M.&Bujard,H.(1995)Efficacyoftetracycline-controlledgeneexpressionisinfluencedbycelltype.BioTechniques89:213–215.
Gossen,M.,Freundlieb,S.,Bender,G.,Muller,G.,Hillen,W.&Bujard,H.(1995)Transcriptionalactivationbytetracyclineinmammaliancells.Science 268:1766–1769.
HarkinD.P.,BeanJ.M.,MiklosD,SongY.H.,TruongV.B.,EnglertC,ChristiansF.C.,EllisenL.W.,MaheswaranS.,OlinerJ.D.,HaberD.A.(1999)InductionofGADD45andJNK/SAPK-dependentapoptosisfollowinginducibleexpressionofBRCA1.Cell 97:575–586.
Hillen,W.&Berens,C. (1994)MechanismsunderlyingexpressionofTn10-encoded tetracyclineresistance.Annual. Rev. Microbiol.48:345–369.
Kozak,M.(1987)Ananalysisof5'-noncodingregionsfrom699vertebratemessengerRNAs.NucleicAcidsRes.15:8125–8148.
Li,X.,Zhao,X.,Fang,Y.,Jiang,X.,Duong,T.,Huang,C.-C.&Kain,S.R.(1998)Generationofdestabilizedenhancedgreenfluorescentproteinasatranscriptionreporter. J. Biol. Chem. 273:34970–34975.
LinearSelectionMarkers(2003)Clontechniques XVIII(2):11.
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3001-1 3� Version No. PR58969
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X. References continued
pTRE-TightVectors(2003)Clontechniques XVIII(2):10–11.
Rennel,E.&Gerwins,P.(2002)Howtomaketetracycline-regulatedtransgeneexpressiongoonandoff.Anal. Biochem.309:79–84.
Resnitzky,D.,Gossen,M.,Bujard,H.&Reed,S.I.(1994)AccelerationoftheG1/SphasetransitionbyexpressionofcyclinsD1andEusinganinduciblesystem.Mol. Cell. Biol.14:1669–1679.
Sambrook,J.,Fritsch,E.F.&Maniatis,T.(2001).Molecular Cloning: A Laboratory Manual,ColdSpringHarborLaboratoryPress(ColdSpringHarbor,NY).
Triezenberg,S.J.,Kingsbury,R.C.&McKnight,S.L.(1988)FunctionaldissectionofVP16,thetrans-activatorofherpessimplexvirusimmediateearlygeneexpression.Genes Devel. 2:718–729.
Witzgall,R.,O'Leary,E.,Leaf,A.,Onaldi,D.&Bonventre,J.V.(1994)TheKruppel-associatedbox-A(KRAB-A)domainofzincfingerproteinsmediatestranscriptionalrepression.Proc Natl Acad Sci USA91:4514–4518.
Yao,F.,Svenjo,T.,Winkler,T.,Lu,M,Eriksson,C.&Eriksson,E.(1998)Tetracyclinerepressor,tetR,ratherthanthetetR-mammaliancelltranscriptionfactorfusionderivatives,regulatesinduciblegeneexpressioninmammaliancells.Hum. Gene Ther.9:1939–1950.
Yarronton,G.T.(1992)Induciblevectorsforexpressioninmammaliancells.Curr. Opin. Biotechnol.3:506–511.
Yin,D.X.&Schimke,R.T.(1995)Bcl-2expressiondelaysdrug-inducedapoptosisbutdoesnotincreaseclonogenicsurvivalafterdrugtreatmentinHeLacells. Cancer Res.55:4922–4928.
Yin,D.X.,Zhu,L.&Schimke,R.T.(1996)Tetracyclinecontrolledgeneexpressionsystemachieveshigh-levelandquantitativecontrolofgeneexpression.Anal. Biochem.235:195–201.
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Tet Systems User Manual
XI. Related Products
ForacompletelistingofallClontechproducts,pleasevisitwww.clontech.com
Cat. No.
• Tet-OffandTet-OnCellLines many Response Vectors • pBIBidirectionalTetVector 631006 • pBI-GBidirectionalTetVector 631004 • pBI-LBidirectionalTetVector 631005 • pTRE2Vector 631008 • pTRE-MycVector 631010 • pTRE2hyg2-MycVector 631052 • pTRE2pur-MycVector 631055 • pTRE-HAVector 631012 • pTRE2hyg2-HAVector 631051 • pTRE2pur-HAVector 631054 • pTRE-6xHNVector 631009 • pTRE2hyg2-6xHNVector 631053 • pTRE2pur-6xHNVector 631056 • pTRE2hygVector 631014 • pTRE2purVector 631013 • pTRE-TightVector 631059 • pTRE-Tight-DsRed2Vector 631061 • pLP-TRE2AcceptorVector 631016Regulator Vectors • pTet-OffVector 631017 • pTet-OnVector 631018 • VP16MinimalDomainVectorSet 631019 • pTet-tTSVector 631011Selection Markers • pTK-HygVector 631750 • pPURVector 631601 • LinearHygromycinMarker 631625 • LinearPuromycinMarker 631626Sequencing Primers • pTRESequencing/PCRPrimers 631104 • pTRE2Sequencing/PCRPrimers 631103
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3001-1 3� Version No. PR58969
Tet Systems User Manual
XI. Related Products continued
Cat. No. Retroviral Expression Systems • RevTet-Off™System 631020 • RevTet-On™System 631021 • RevTet-Off™SystemwithCreator™Technology 631023 • RevTet-On™SystemwithCreator™Technology 631024 • pRevTet-OffVector 631003 • pRevTet-OnVector 631007 • pRevTet-Off-INVector 631001 • pRevTREVector 631002 • pLP-RevTRE 631015 • PackagingCellLines manyAdenoviral Expression Systems • Adeno-X™Tet-OffSystem 631022 • Adeno-X™Tet-OnSystem 631050 • Adeno-X™Tet-OffSystem2 631058 • Adeno-X™Tet-OnSystem2 631057Cell Culture • TetSystemApprovedFBS,US-Sourced 631101 • TetSystemApprovedFBS,USDA-Approved 631106 • CalPhos™MammalianTransfectionKit 631312 • CLONfectin™TransfectionReagent 631301 • G418 631307 • HygromycinB 631309 • Doxycycline 631311 • Puromycin 631305 • Anhydrotetracycline 631310Other Related Products • NucleoBond®andNucleoSpin®Columns many • NucleoTrap®GelExtractionkit 636018 • NucleoTrap®PCRPurificationKit 636020 • LuciferaseReporterAssayKit 631714 • Creator™pDNRCloningKits 631615 • pCMVβVector 631719 • pAcGFP-N1Vector 632469 • pAcGFPVectorSet 632426
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Tet Systems User Manual
Table I. Tet-Off and Tet-On Vector alignment.
Appendix A: Vector Information
gene of interest
gene of interestgene of interest
gene of interest
luc
lacZPminCMV PminCMVTRE
PminCMV PminCMVTRE
PminCMV PminCMVTRE
Response vectors for monitoring expression of a target gene via expression of a coregulated reporter
Bidirectional Tet Vectors
pBI-G
pBI-L
pBI-Tet
poly Agene of interest
poly A HygR/PurRgene of interest
TRE PminCMV
tetRPCMV
rtetR VP16PCMV
PCMV
TRE PCMV
VP16
poly Agene of interestPminCMVPminCMVTREmod
Regulator vector for use in Tet-Off system
Regulator vector for use in Tet-On system
Response plasmids encoding the Tet Responsive Element (TRE)for use in either Tet-Off or Tet-On
Name Applications
pTet-Off
pTet-On
pTRE2
pTRE2hyg/pur
pTRE-Tight
Basic Vectors
c-myc gene of interest poly A
HA gene of interest poly A
6xHN gene of interest poly A
PminCMVTRE PCMV
TRE PCMV
TRE PCMV
Response plasmids for use in either Tet-Off or Tet-On System Used for screening with antibodies or for purification
Tagged Vectors
pTRE-Myc
pTRE-HA
pTRE-6xHN
Response plasmid encoding amodified Tet Responsive Element (TREmod)for use in either Tet-Off or Tet-On
tetR SDkid-1 poly A
tetR VP16-2, 3, 4PCMV
PCMV
Minimal domain vectors used inTet-Off System; minimizes VP16 toxicity
For tighter control of gene expression in Tet-On SystemspTet-tTS
ptTA-2, 3, 4
pTRE-Tight-DsRed2
Accessory Vectors
poly ADsRed2PminCMV Reporter vector containing amodified Tet Responsive Element (TREmod)for use in either Tet-Off or Tet-On
TREmod
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Tet Systems User Manual
Appendix A: Vector Information continued
Table I. Tet-Off and Tet-On Vector alignment continued
gene of interest 3' LTR�' LTR Ψ+
tetR 3' LTR�' LTR VP1� IRESΨ+
3' LTRtetR VP1��' LTR Ψ+
NeoR
3' LTR rtetR VP1��' LTR Ψ+ NeoR
NeoR
HygR
PCMV
PCMV
TRE PminCMV
RevTet Basic Vectors
Can be used for quickly establishing a Tet-Off cell line
pRevTet-Off-IN
pRevTet-Off
pRevTet-On
pRevTRE
RevTet Accessory Vectors
Regulator vector for use in RevTet-Off System
Regulator vector for use in RevTet-On SystemResponse vector for use in eitherRevTet-Off or RevTet-On Systems
Name Applications
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Tet Systems User Manual
Appendix A: Vector Information continued
TAB
LE
II: T
ET
SY
ST
EM
S V
EC
TOR
INF
OR
MA
TIO
N
Mam
mal
ian
Dia
gn
ost
ic
Fra
gm
ent
Nam
e in
E
xpre
ssed
se
lect
able
S
ize
rest
rict
ion
si
zes
N
ame
Ref
eren
ce
refe
ren
ce
pro
tein
m
arke
r (K
b)
enzy
me(
s)
(kb
)
pTet
-Off
Res
nitz
kye
t al.
(19
94)
pUH
D15
-1ne
otT
A
neom
ycin
7.
37
Xho
I&
2.
9,2
.2,
V
ecto
rG
osse
n&
Buj
ard
(199
2)
Hin
dIII
2.
3
pTet
-On
G
osse
net
al.
(19
95)
pUH
D17
-1ne
ort
TA
neom
ycin
7.
37
Xho
I&
2.
9,2
.2
V
ecto
r
Hin
dIII
1.
5,0
.9
pTet
-tT
S
Fre
undl
ieb
et a
l. (
1999
)pU
HS
6-1
tT
S
none
4.
3E
coR
I&
3.
0,1
.3
Vec
tor
H
ind
IIIpT
RE
2hyg
pr
otei
nX
hy
grom
ycin
5.
3X
hoI
3.75
,1.5
5V
ecto
r
Bam
HI
5.3
pTR
E2h
yg-L
uc
luci
fera
se
hygr
omyc
in
6960
X
hoI
5.4,
1.5
5(c
ontr
olp
rovi
ded
with
pT
RE
2hyg
)
pTR
E2p
ur
prot
ein
X
puro
myc
in
5.1
Xho
I5.
4,1
.55
Vec
tor
B
amH
I5.
1pT
RE
2pur
-Luc
lu
cife
rase
pu
rom
ycin
6.
73
Xho
I5.
4,1
.55
(c
ontr
olp
rovi
ded
with
pT
RE
2pur
)
B
amH
I4.
5,1
.34
pTR
E2
pr
otei
nX
no
ne
3.76
E
coR
I
3.0,
0.7
Vec
tor
pT
RE
2-Lu
c
luci
fera
se
none
54
06
Eco
RI
3.0,
1.7
,0.7
(c
ontr
olp
rovi
ded
with
pT
RE
2)
pT
RE
-Tig
ht
prot
ein
X
none
2.
6X
hoI
2.0
&0
.6
Vec
tor
pT
RE
-Tig
ht-L
uc
lu
cife
rase
no
ne
4.2
Bam
HI
&
2.6
&1
.6
(con
trol
pro
vide
dw
ithp
TR
E-T
ight
)
N
heI
pTR
E-M
yc
Myc
-pro
tein
X
none
38
38
Eco
RI
3.0,
0.8
V
ecto
rpT
RE
-Myc
-Luc
M
yc-lu
cife
rase
no
ne
5.5
Bam
HI
5.5
(c
ontr
olp
rovi
ded
with
pT
RE
-Myc
)
H
ind
III
1.6,
3.8
pT
RE
2hyg
2-M
yc
M
yc-p
rote
inX
hyg
rom
ycin
5.
4H
ind
III
3.6
&1
.8
E
coR
V
5.4
pTR
E2p
ur-M
yc
Myc
-pro
tein
Xp
urom
ycin
5.
2H
ind
III
3.6
&1
.6
E
coR
V
5.2
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3001-1 �0 Version No. PR58969
Tet Systems User Manual
Appendix A: Vector Information continued
TAB
LE
II: T
ET
SY
ST
EM
S V
EC
TOR
INF
OR
MA
TIO
N c
on
tin
ued
Mam
mal
ian
Dia
gn
ost
ic
Fra
gm
ent
Nam
e in
E
xpre
ssed
se
lect
able
S
ize
rest
rict
ion
si
zes
N
ame
Ref
eren
ce
refe
ren
ce
pro
tein
m
arke
r (K
b)
enzy
me(
s)
(kb
)
pTR
E-H
A
HA
-pro
tein
X
none
3.
83
Bam
HI
3.8
V
ecto
r
Mlu
Ino
tcut
pTR
E2h
yg2-
HA
HA
-pro
tein
X
hygr
omyc
in
5.4
Hin
dIII
3.
6&
1.8
Vec
tor
E
coR
V
5.4
pTR
E2p
ur-H
A
HA
-pro
tein
X
puro
myc
in
5.2
Hin
dIII
3.
6&
1.6
Vec
tor
E
coR
V
5.2
pTR
E-H
A-L
uc
HA
-luci
fera
se
none
54
89
Bam
HI
5.6
(c
ontr
olp
rovi
ded
with
pT
RE
-HA
)
H
ind
III
3.8,
1.8
pTR
E-6
xHN
6x
HN
-pro
tein
X
none
3.
8B
amH
I3.
8V
ecto
r
Eco
RI
3.
0,0
.8
pTR
E2h
yg2-
6xH
N
6x
HN
-pro
tein
X
hygr
omyc
in
5.4
Hin
dIII
3.
6&
1.8
Vec
tor
E
coR
V
5.4
pTR
E2p
ur-6
xHN
6xH
N-p
rote
inX
pu
rom
ycin
5.
2H
ind
III
3.6
&1
.6V
ecto
r
Eco
RV
5.
2pT
RE
-6xH
N-L
uc
6x
HN
-luci
fera
se
none
5.
5H
ind
III
3.8,
1.8
(c
ontr
olp
rovi
ded
with
pT
RE
-6xH
N)
Bam
HI
5.6
pTR
E-T
ight
-DsR
ed2
D
sRed
2no
ne
3.3
Not
I2.
6,0
.7
Vec
tor
B
amH
I
Protocol No. PT3001-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR58969 �1
Tet Systems User Manual
TAB
LE
II: T
ET
SY
ST
EM
S V
EC
TOR
INF
OR
MA
TIO
N c
on
tin
ued
Mam
mal
ian
Dia
gn
ost
ic
Fra
gm
ent
Nam
e in
E
xpre
ssed
se
lect
able
S
ize
rest
rict
ion
si
zes
N
ame
Ref
eren
ce
refe
ren
ce
pro
tein
m
arke
r (K
b)
enzy
me(
s)
(kb
)
pBI
Bar
one
t al.
(19
95)
pB
I-4
prot
ein
X,
none
4.
36
Xba
I3.
7,0
.7
V
ecto
r
pr
otei
nY
pBI-
G
Bar
one
t al.
(19
95)
pB
I-3
β-ga
l,no
ne
7.73
X
baI
3.7,
3.5
,
VE
ctor
pr
otei
nX
0.6
pBI-
LB
aron
et a
l. (
1995
)
pBI-
2lu
cife
rase
,no
ne
6.08
X
baI
3.7,
2.4
Vec
tor
prot
ein
X
pBI-
GL
Bar
one
t al.
(19
95)
pB
I-1
β-ga
l,no
ne
9.5
Xba
I3.
7,3
.5,
(c
ontr
olp
rovi
ded
with
pB
I,pB
I-G
,and
pB
I-L)
luci
fera
se
2.
3pT
K-H
yg
none
hy
grom
ycin
5.
07
Eco
RI
0.4,
1.0
,1.3
,2.3
V
ecto
r
Appendix A: Vector Information continued
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3001-1 �� Version No. PR58969
Tet Systems User Manual
Appendix A: Vector Information continued
AnannotatedprintoutofthepTRE2hygsequence(PT3521-5)isprovidedwiththeTet-OffandTet-OnGeneExpressionSystems.Youcanobtainthesequencesoftheothervectorsatwww.clontech.com.
Figure 9. pTet-Off and pTet-On composite vector map. Uniquesitesareinbold.OnlypTet-OncontainsthesecondHindIIIsiteatPositionNo.871.ThissitecanbeusedtodistinguishpTet-OfffrompTet-On.pTet-OffexpressesthetTA(tettransactivator)regulatorproteinfromthestrongimmediateearlypromoterofcytomegalovirus(PCMV).pTet-OnexpressesthertTA(reversetTA),whichcontainsfouramino-acidmutations(asmarkedonthemap).Inaddition,thereareseveralsilentmutationsinpTet-On.Inallotherrespects,thevectorsareidentical.
D60167pr.bw/pTet-OffDRAFT3/1/96
Hind III(2250)
Xho I(2)
pTet-OffpTet-On
�.� kb
Col E1ori
Ampr
PCMV
SV40poly A
Neor
Xho I(4461)
Bsa I(3546)
Sca I(3949)
VP16AD
(r)tetR
Hind III (pTet-On only)(871)
(r)tTA
= Mutations that convert TetR to rTetR (and tTA to rtTA)
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Tet Systems User Manual
Appendix A: Vector Information continued
Figure 10. pTRE2hyg and pTRE2pur vector maps and MCSs. BothresponsevectorscontainanMCSimmediatelydownstreamoftheTet-responsivePhCMV*-1promoter.PhCMV*-1containstheTetresponseelement(TRE),whichconsistsofsevencopiesofasequencecontainingthe19-bptetoperatorsequence(tetO),andtheminimalCMVpromoter(PminCMV),whichlackstheenhancerthatispartofthecompleteCMVpromoterintheregulatoryplasmids.Consequently,PhCMV*-1issilentintheabsenceofbindingofTetRorrTetRtothetetOsequences.GenesinsertedintooneofthesitesintheMCSwillberesponsivetothetTAandrtTAregulatoryproteinsintheTet-OffandTet-Onsystems,respectively.NotethattheclonedinsertmusthaveaninitiatingATGcodon.TheadditionofaKozaksequenceisnotrequired,butmayimproveexpressionlevels.Theadditionofaninternalselectionelement(HygrorPuror)eliminatestheneedforcotransfectionwithpTK-Hyg.Completesequenceinformation isprovided inthepTRE2hygandpTRE2purVector InformationPackets(pTRE2hyg:PT3521-5;pTRE2pur:PT3520-5).
pTRE2hyg-Luc and pTRE2pur-Luccontain thegeneencodingfirefly luciferasecloned into theBamHIandNheIsitesinthepTRE2hygandpTRE2purMCS.TheNheIsitesweredestroyedduringconstruction.Theluciferaseconstructadds1,649bptothevectors.
514•
GGATCCTCTAGTCAGCTGACGCGTGCTAGCGCGGCCGCATCGAT
AAGCTTGTCGACGATATCTCTAGA
BamH I Pvu II
EcoR V
Mlu I Nhe I Cla INot I
470•
480•
490•
500•
510•
520•
530•
pTRE�pur�.1 kb
Col E1ori
Ampr
TRE
β-globinpoly A
Xho I(2)
Pvu I(3142)
PhCMV*-1
PminCMV
MCS(470-537)
Xho I(3759)
Puror
EcoR I(450)
EcoR I(1175)
PSV40
SV40poly A
514•
GGATCCTCTAGTCAGCTGACGCGTGCTAGCGCGGCCGCATCGAT
AAGCTTGTCGACGATATCTCTAGA
BamH I Pvu II
EcoR VSal IAcc I
Mlu I Nhe I Cla INot I
470•
480•
490•
500•
510•
520•
530•
pTRE�hyg�.3 kb
Col E1ori
Ampr
TRE
β-globinpoly A
Xho I(2)
PhCMV*-1
PminCMV
MCS(470–537)
Xho I(3759)
Hygr
SV40poly A
PSV40
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3001-1 �� Version No. PR58969
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Appendix A: Vector Information continued
pTRE-Tight2.6 kb
Col E1ori
Ampr
TREmod
SV40poly A
Xho I(2)
Pvu I(1985)
Ptight
PminCMV
MCS(323– 411)
Xho I(602)
Figure 11. pTRE-Tight vector map and MCS. This response plasmid contains an MCSimmediatelydownstreamoftheTet-responsivePtightpromoter.PtightcontainsamodifiedTetresponseelement(TREmod),whichconsistsofsevendirectrepeatsofa36-bpsequencethatcontainsthe19-bp tet operator sequence (tetO) and the minimal CMV promoter (PminCMV∆), which lacks theenhancerthatispartofthecompleteCMVpromoter.Consequently,PtightissilentintheabsenceofbindingofTetRorrTetRtothetetOsequences.GenesinsertedintotheMCSwillberesponsivetothetTAandrtTAregulatoryproteinsintheTet-OffandTet-Onsystems,respectively.NotethattheclonedinsertmusthaveaninitiatingATGcodon.TheadditionofaKozaksequenceisnotrequired,butmayimproveexpressionlevels.pTRE-Tight-GeneXplasmidsshouldbecotransfectedwiththeLinearHygromycinMarker(Cat.No.631625,notincluded)orLinearPuromycinMarker(Cat.No.631626,notincluded)topermitselectionofstabletransfectants.CompletesequenceinformationisprovidedinthepTRE-TightVectorInformationPacket(PT3720-5).ThepTRE-Tight-LucControlVector,packagedwiththepTRE-TightVector,containsanadditional1,649bpencodingfireflyluciferaseinsertedintotheMCS.Thisvectorcanbeusedasareporterofinductionefficiency.Itisnotintendedasacloningvector.pTRE-Tight-DsRed2 containsthegeneencodingDsRed2clonedintotheBamHIandNotIsitesinthepTRE-TightMCS.DsRed2isavariantoftheredfluorescentproteinisolatedfromtheIndoPacificseaanemonerelativeDiscosoma sp.
GAATTCGAGCTCGGTACCCGGGGATCCTCTAGTCAGCTGACGCGT
GCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTCTAGA
323•
368•
BamH IKpn I Pvu II Mlu I
Nhe I Eag I Cla I Hind III Xba IEcoR VSal IAcc INot I
EcoR I Sac ISma I
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Appendix A: Vector Information continued
Figure 12. pTRE-Myc, -HA and -6xHN composite vector map and multiple clone site (MCS).ThesetaggedpTREvectorscontainanMCSimmediatelydownstreamoftheTet-responsivePhCMV*-
1promoter.PhCMV*-1containstheTetresponseelement(TRE),whichconsistsofsevencopiesofa sequencecontaining the19-bp tetoperator sequence (tetO), and theminimalCMVpromoter(PminCMV),whichlackstheenhancerthat ispartof thecompleteCMVpromoter intheregulatoryplasmids.Consequently,PhCMV*-1 issilent in theabsenceofbindingofTetRorrTetRto the tetOsequences.GenesinsertedintooneofthesitesintheMCSwillberesponsivetothetTAandrtTAregulatoryproteinsintheTet-OffandTet-Onsystems,respectively.NotethattheclonedinsertmustbeinframewiththetagandneednothaveanATGorKozaksequence,astheseareprovidedatthestartofthetag.Thetaggedfusionproteincanbeefficientlydetectedandpurifiedusingantibodiesand resins optimized against the different markers. Complete sequence information is providedinthepTRE-Myc,-HA,and-6xHNVectorInformationPackets(pTRE-Myc:PT3398-5;pTRE-HA:PT3462-5;pTRE-6xHN:PT3463-5).
PhCMV*-1
Pmin CMV
pTRE-Myc,-HA & -�xHN
3.� kb
Col E1ori
Ampr
TRE
β-globinpoly A
MCS
tag
GAATTCGAGCTCGGTACCCGGGGATCCTCTAGTCAGCTGACGCGT
GCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTCTAGA
323•
368•
BamH IKpn I Pvu II Mlu I
Nhe I Eag I Cla I Hind III Xba IEcoR VSal IAcc INot I
EcoR I Sac ISma I
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Figure 13. pTRE2hyg2-Myc, -HA & -6xHN and pTRE2pur-Myc, -HA & -6xHN composite vector map and multiple clone site (MCS).ThesetaggedpTREvectorscontainaproteintagsequencefollowed by an MCS immediately downstream of theTet-responsive PhCMV*-1 promoter. PhCMV*-1containstheTetresponseelement(TRE),whichconsistsofsevencopiesofasequencecontain-ingthe19-bptetoperatorsequence(tetO),andtheminimalCMVpromoter(PminCMV),whichlackstheenhancerthatispartofthecompleteCMVpromoterintheregulatoryplasmids.Consequently,PhCMV*-1 issilent intheabsenceofbindingoftTAorrtTAtothe tetOsequences.GenesinsertedintooneofthesitesintheMCSwillberesponsivetothetTAandrtTAregulatoryproteinsintheTet-Off andTet-On systems, respectively.The tagged fusion protein can be efficiently detectedand purified using antibodies and resins optimized against the different markers. Complete se-quence information is provided in the Vector Information Packets (pTRE2hyg2-Myc: PT3685-5;pTRE2hyg2-HA: PT3684-5; pTRE2hyg2-6xHN: PT3686-5; pTRE2pur-Myc: PT3688-5;pTRE2pur-HA:PT3687-5;pTRE2pur-6xHN:PT3689-5).
Appendix A: Vector Information continued
ATG XXX XXX XXX XXX XXX XXX XXX XXX XXX XXX XXX XXX XXX CTT ATG GCC ACT
GAC GCG TTG CTA GCG CAG CTG GAA GCT TAT CGA TTG CGG CCG CGT CGA CGA TAT C
504•
Mlu I Not I EcoR VNhe I Cla I
c-Myc, HA or 6xHN epitope tag
pTRE�Marker-Myc,-HA & -�xHN
Col E1ori
Ampr
TRE2
β-globinpoly A
PhCMV*-1
PminCMV
MCS
Purr/Hygr
SV40poly A
PSV40
= tag sequence
Protocol No. PT3001-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR58969 ��
Tet Systems User Manual
Appendix A: Vector Information continued
pTK-Hyg�.1 kb
pUCori
Ampr
HSV TKpoly A
Hygr
PHSV TK
EcoR I (2718) EcoR I (2330)
EcoR I (1017)
EcoR I (5040)
Figure 14. pTK-Hyg plasmid map. pTK-Hyg is cotransfected with pTRE-derived plasmids(but not withpTRE2hyg andpTRE2purvectors)toallowselectionofstablytransformedcelllinesin thepresenceofhygromycin.TheabsenceofanenhancerelementonpTK-Hygprevents theunwantedactivationofpTRE-derivedplasmidsuponcointegrationintothegenome.ThesequenceofpTK-HyghasbeendepositedinGenBank(AccessionNo.U40398).
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3001-1 �� Version No. PR58969
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Figure 15. The pBI expression cassette.Twogenes—eithertwogenesofinterest,ageneofinterestandareporter,ortworeporters—canbeexpressedsimultaneouslyfromthePbipromoter.Reportersarefireflyluciferase,orβ-galactosidase.
Bidirectional (pBI) Tet VectorsTheBidirectionalTetVectorsareused to simultaneouslyexpress twogenesunder control ofa singleTRE (Baronet al., 1995; formore information, seeClontechniques,October1996,p.8).AfteraTet-OfforTet-Oncelllineisestablished,apBIvectoriscotransfectedwithpTK-Hygtopermitselectionofadouble-stabletet-responsivecelllinethatcoexpressestwogenes.pBI-G, and pBI-L can be used to indirectly monitor expression of a gene ofinterestforwhichthereisnodirectorconvenientassay.Thesevectorsexpress β-galactosidase,orluciferaseasthereportergenelocatedononesideoftheTRE.GeneXcanbeexpressedatthesametimeasthereporterwhenclonedintotheMCSflankingtheothersideoftheTRE.Whenscreeningdouble-stablecell lines(SectionIX.D),youcanmonitorexpressionof thereporter fromthevector that also simultaneously expresses the gene of interest. ExpressionlevelsofthegeneofinterestcanbeinferredfromreportergeneexpressioninresponsetoTcorDox.ThepBIVector lacks reporter sequencesand insteadcontains twoseparateMCSsinoppositeorientationdrivenbytwoidentical,induciblepromoters.pBIallowsforcoexpressionoftwogenesofinterestinthesamecell.Forinstance,the interaction of two proteins or two subunits of a complex protein can beinvestigatedbysimultaneousexpressioninpBI.Visitwww.clontech.com forcompletevectorinformation.
Appendix A: Vector Information continued
PminCMV-1TREPminCMV-�
Pbi-1
Gene X Reporter
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Appendix A: Vector Information continued
Figure 16. VP16 Minimal Domain Vectors.ThethreevectorsdifferinthesequenceoftheirVP16activationdomains.The letters in thefirstcolumnofPanelB indicate theaminoacidat thekeyfunctionalpositionofa13aminoacidrepeatthatcomposestheminimaldomains.TherestofthevectorisidenticaltopTet-Off.TheactivationdomainfromeachvectoristoleratedatdifferentlevelsandcausesactivationatdifferentlevelsrelativetopTet-Off(PanelB).nd=notdetermined.
A
B
F
F F F
FF
G F Y
ptTA2
ptTA3
pTet-Off
ptTA4
Stableregulation
factor
Toleratedlevel of
activator
Relativetransient
activation (%)
1X 100 2.2 x 105
3X 98 nd
5X 39 1.5 x 105
9X 14 4.4 x 104
VP16 activation domain
ptTA�/3/��.1 kb
Ampr
PCMV
SV40poly A
Neor
tTAVP16 minimal
activation domain
tetR
Col E1ori
Hind III(1987)
Bsa I(3267)
Sca I(3686)
VP16 Minimal Domain Vectors
TheVP16MinimalDomainVectorSet(Cat.No.631019)—ptTA2,ptTA3,andptTA4—expressestetracycline-controlledtransactivatorscontainingmodifiedVP16activationdomains(Figure17;Baronet al.,1997).OverexpressionofunmodifiedVP16canhavenegativepleiotropiceffectsdue to interactionswithessentialcomponentsofthetranscriptionalmachinery.Thisgenerallydoesnotinterferewithin vitroexpression,butcanposeproblems in vivowhentTAtranscriptionisdrivenbyastrongtissue-specificpromoter.
The modified VP16 moieties contained in these transactivators allows theirexpressionathigherintracellularlevels,potentiallyallowingincreasedstabilityforcellcultureandtransgenicapplications(Baronet al.,1997).Furthermore,eachvectorallowsproteinexpressionoveradifferent inductionrange(PanelB).Applicationssuchas knock-in/knock-outexperiments relyonsite-specificintegrationandthusaredependentonthetranscriptionalactivityoftheparticularlocus.Inthesesituations,theVP16MinimalDomainVectorsmayenableyoutoobtainoptimalexpression levelsbyadaptingtheactivationpotentialof thetransactivatortotheexpressionlevelofthelocus.
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Appendix A: Vector Information continued
The pTet-tTS VectorThepTet-tTSVector(Cat.No.631011)isdesignedforusewiththeTet-OnSystem.ItisnotsuitableforusewiththeTet-OffSystem.pTet-tTSpreventsunregulatedgeneexpressionintheabsenceofDox(Clontechniques,April1999).Itexpressesthe tetracycline-controlled transcriptional silencer (tTS), which is a fusion ofTetRand theKRAB-ABdomainof theKid-1protein (Freundliebet al.,1999;Witzgallet al. ,1994).IntheabsenceofDox,tTSbindsthetetOsequenceintheTREandactivelysilencestranscriptionofGeneX(Figure18).AsDoxisaddedtotheculturemedium,thetTSdissociatesfromtheTRE,relievingtranscriptionalsuppression.AtsufficientconcentrationsofDox,thertTAtransactivatorbindstheTREandactivatestranscriptionofGeneX.ForadditionalinformationonpTet-tTS,includingavectormap,pleaserefertothepTet-tTSVectorInformationPacket(PT3334-5),availableatwww.clontech.com.
Figure 17. Dose response curve demonstrating controlled expression in a cell line coexpressing tTS and rtTA. HR5cells,whichconstitutivelyexpressrtTA,weretransientlytransfectedwithaplasmidexpressingtTSandacontrolvectorexpressingluciferasedownstreamoftheTRE.CellswereculturedintheindicatedlevelsofDox.After24hr,cellswereharvestedandassayedforluciferaseactivity.SD=silencingdomain.AD=activationdomain.DataprovidedcourtesyofS.Freundlieb,ZentrumfürMolekulareBiologie(ZMBH),UniversitätHeidelberg.
0 1 10 100 1,000 10,0000
2
4
6
8
10
12
Doxycycline (ng/ml)
Arb
itrar
y lig
ht u
nits
TRE TATA
TRE TATA
TetRTRE TATA
AD
TetRSD
X
rtTA
tTS
TetRSD
No transcription
Induced high transcription
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Tet Systems User Manual
Appendix B: Glossary
Dox Doxycycline,aderivativeofTc that is thepreferredeffectorsubstanceforTetexperiments.
Double-stable ATet-OfforTet-OncelllinethathasbeenstablytransfectedTetCellLine withpTRE2-GeneXconstruct.GeneXisinducedbytheremoval (forTet-Off)oraddition(forTet-On)ofDoxfromthemedia.
GeneX Thegeneofinterest,clonedintotheResponsePlasmid.
PCMV Thecompleteimmediateearlypromoterofcytomegalovirus.Thisisaprovenstrongpromoterinmanymammaliancelltypes.
PminCMV Theminimal immediateearlyCMVpromoter.ThispromoterlacksthestrongCMVenhancer,andisthereforesilentintheabsenceofbindingoftTAorrtTAtotheTRE.
PminCMV∆ AnalteredminimalimmediateearlyCMVpromoter.ThispromoterisusedinthepTRE-Tightvectorseries.
PhCMV*-1 ThecompoundpromoterinpTREandrelatedvectorsthatconsistsoftheTREelementlocatedjustupstreamofPminCMV.
Ptight ThecompoundpromoterinthepTRE-TightvectorsthatconsistsoftheTREmodelementlocatedjustupstreamofPminCMV∆.
Regulator The plasmid that encodes the hybrid regulatory proteinPlasmid (tTAorrtTA)inaTet-OfforTet-OnSystem–i.e.,pTet-OfforpTet.
Response ApTRE-derivedplasmidthatexpressesageneofinterestfromPlasmid thePhCMV*-1promoter.ApTRE-derivedplasmidcanbeusedin
bothTet-OffandTet-Onsystems.
rTetR ThereverseTetrepressor.InE. coli,rTetRbindsspecificallytotetOandblockstranscriptionofthetetoperoninthepresenceofTc.
rtTA Reversetetracycline-controlledtransactivator:A37kDafusionproteinconsistingoftherTetRandtheVP16activationdomain(AD).BindsspecificallytoTREandactivatestranscriptioninthepresenceofDox.
Tc Thechemicalcompoundtetracycline
Tet Tetracycline, as in the tet operon or the Tet repressor.(ThecompoundtetracyclineisabbreviatedTc.)
Tet-Off AnycelllinethatstablyexpressestTAfromintegratedcopiesCellLines ofpTet-Off.Tet-Offcelllinescaneitherbemadebytheresearcher
orpurchasedfromClontech.
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Appendix B: Glossary continued
Tet-On AnycelllinethatstablyexpressesrtTAfromintegratedcopiesCellLines ofpTet-On.Tet-Oncelllinescaneitherbemadebytheresearcher
orpurchasedfromClontech.
tetO Thetetoperator,a19-bp,cis-actingregulatoryDNAsequencefromthebacterialtetoperon,whereitisthenaturalbindingsiteforTetR.SeeTRE.
TetR TheTetrepressorcomponentoftTAandrtTA.InE. coli,TetRbinds specifically to tetO and blocks transcription of the tetoperonintheabsenceofTc.
TRE Tet-ResponseElement.Aregulatorysequenceconsistingofsevendirect repeatsofa42-bpsequence thatcontains thetetO.
TREmod Modified Tet-Response Element. A regulatory sequenceconsistingofsevendirectrepeatsofa36-bpsequencethatcontainsthetetO.
tTA Tetracycline-controlledtransactivator:A37kDafusionproteinconsistingoftheTetRandtheVP16activationdomain(AD).BindsspecificallytotheTREandactivatestranscriptionintheabsenceofTcorDox.
tTS Tetracycline-controlledtranscriptionalsilencer,afusionproteinconsisting of the TetR and the KRAB-AB domain of Kid-1.BindsspecificallytotheTREandsuppressestranscriptionintheabsenceofDox.
VP16AD TheactivationdomainoftheVP16proteinfromherpessimplexvirus.
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Notes
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Notes
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Tet Systems User Manual
Notes
Notice to Purchaser
Clontechproductsaretobeusedforresearchpurposesonly.Theymaynotbeusedforanyotherpurpose,including,butnotlimitedto,useindrugs,invitrodiagnosticpurposes,therapeutics,orinhumans.Clontechproductsmaynotbetransferredtothirdparties,resold,modifiedforresale,orused tomanufacturecommercialproductsor toprovideaservice to thirdpartieswithoutwrittenapprovalofClontechLaboratories,Inc.
Use of theTetracycline controllable expression systems (the "TetTechnology") is covered by aseries of patents including U.S.PatentNos. 5,464,758and 5,814,618,whichareproprietary toTETSystemsHoldingGmbH&Co.KG.Academicresearchinstitutionsaregrantedanautomaticlicense with the purchase of this product to use theTetTechnology only for internal, academicresearchpurposes,whichlicensespecificallyexcludestherighttosell,orotherwisetransfer,theTetTechnologyoritscomponentpartstothirdparties.Inacceptingthislicense,allusersacknowledgethattheTetTechnologyisexperimentalinnature.TETSystemsHoldingGmbH&Co.KGmakesnowarranties,expressorimpliedorofanykind,andherebydisclaimsanywarranties,representations,orguaranteesofanykindastotheTetTechnology,patents,orproducts.AllothersareinvitedtorequestalicensefromTETSystemsHoldingGmbH&Co.KGpriortopurchasingthesereagentsorusingthemforanypurpose.Clontechisrequiredbyitslicensingagreementtosubmitareportofallpurchasersof theTet-controllableexpressionsystemto IPMerchandisers, Inc.For licenseinformation,pleasecontact:
HansPeterKneubuehlTETSystemsHoldingGmbH&Co.KGImNeuenheimerFeld58269120HeidelbergGermany
Tel+49(0)62215880400Fax+49(0)62215880404E-mail:kneubuehl@tet-systems.deWeb:www.tetsystems.com/licensing
ThisproductisalsosoldunderpatentsublicenseFORRESEARCHPURPOSESONLY.LicensesforcommercialmanufactureoruseunderthispatentmaybeobtaineddirectlyformHarvardUniversity.
NucleoBond®andNucleoSpin®areregisteredtrademarksofMACHEREY-NAGELGmbH&Co.
FLIPR®isaregisteredtrademarkofMolecularDynamicsCorporation
Clontech,ClontechLogoandallothertrademarksarepropertyofClontechLaboratories,Inc.ClontechisaTakaraBioCompany.©2006
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