tet-off and tet-on gene expression systems - …16].pdf · 2017-09-12 · published 13 september...

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

Protocol No. PT3001-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR58969 3


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



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 –

Protocol No. PT3001-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR58969 �


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)




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




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,




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).




• 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).

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3001-1 10 Version No. PR58969


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.





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).

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3001-1 1� Version No. PR58969


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.



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



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)

Protocol No. PT3001-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR58969 1�


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)



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.



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



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.



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.

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3001-1 �0 Version No. PR58969


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.

Protocol No. PT3001-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR58969 �1


VII. Pilot Experiments

5 x 103

10 x 103

15 x 103

Fold

-ind

uctio

n

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.

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3001-1 �� Version No. PR58969


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.




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

0

500

1000

1500

2000

2500

3000

Luci

fera

se a

ctiv

ity (R

LU)

0 0.1 1.0 10 100 1000

Doxycycline (ng/ml)

.001 .01101

102

103

104

105

106

107

.1 1 10 100 1000 10000

Luci

fera

se a

ctiv

ity (a

rbitr

ary

units

)lo

g tr

ansf

orm

ed

Doxycycline (ng/ml)

CHO-AA�-Luc Tet-Off Control CellsA B



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.


Protocol No. PT3001-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR58969 ��


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.



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



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.



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.


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.



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

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3001-1 30 Version No. PR58969


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.


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).




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.




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.



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.



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.



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



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



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



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




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




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



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





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)




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




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




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



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).


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




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).



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.


PminCMV-1TREPminCMV-�

Pbi-1

Gene X Reporter




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.




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



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.



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.



Notes



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:[email protected]:www.tetsystems.com/licensing

ThisproductisalsosoldunderpatentsublicenseFORRESEARCHPURPOSESONLY.LicensesforcommercialmanufactureoruseunderthispatentmaybeobtaineddirectlyformHarvardUniversity.

NucleoBond®andNucleoSpin®areregisteredtrademarksofMACHEREY-NAGELGmbH&Co.

FLIPR®isaregisteredtrademarkofMolecularDynamicsCorporation

Clontech,ClontechLogoandallothertrademarksarepropertyofClontechLaboratories,Inc.ClontechisaTakaraBioCompany.©2006

tet-off and tet-on gene expression systems - …16].pdf · 2017-09-12 · published 13 september...

Documents