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Pathway Profiling System User Manual

PT3286-1 (PR15835)Published 05/02/2001

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3286-1 2 Version No. PR15835


Table of Contents

I. Introduction 4

II. Pathway Profiling Vectors 7

III. Products for Signal Transduction Research 8

IV. List of Components 9

V. Additional Materials Required 12

VI. General Considerations 13

A. UseofControls 13

B. TransfectionConsiderations 13

C. OptimizationofTransfection 14

VII. Pathway Profiling System Procedure 15

VIII. Troubleshooting Guide 17

IX. References 18

X. Related Products 19

Appendix: Pathway Profiling System Vectors 21

Notice to Purchaser

Clontechproductsaretobeusedforresearchpurposesonly.Theymaynotbeusedforanyotherpurpose,including,butnotlimitedto,useindrugs,in vitrodiagnosticpurposes,therapeutics,orinhumans.Clontechproductsmaynotbetransferredtothirdparties,resold,modifiedforresale,orusedtomanufacturecommercialproductsortoprovideaservicetothirdpartieswithoutwrittenapprovalofClontechLaboratories,Inc.

Certainaspectsofthecis-actingreporterconstructproductsarethesubjectofpendingU.S.patents.CalPhos™, CLONfectin™, Great EscAPe™, are trademarks and Living Colors® is a registeredtrademarkofClontechLaboratories,Inc.NucleoBond®isaregisteredtrademarkofMacherey-NagelGmbHandCo.Clontech,theClontechlogoandallothertrademarksarethepropertyofClontechLaboratories,Inc.ClontechisaTakaraBioCompany.©2006

Protocol No. PT3286-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR15835 3


List of Figures

Figure 1. GeneralschemeforthePathwayProfilingprocedure 5

Figure 2. Anoverviewofthe cis-actingenhancerelementvectorcon-structsinthePathwayProfilingSystems 7

Figure 3. GeneralizedmapofthePathwayProfilingVectors 23

Figure 4. MapandmultiplecloningsitesofpTAL-SEAP/LucVector 24

Figure 5. MapandmultiplecloningsitesofpTA-SEAP/LucVector 25

Figure 6. MapandmultiplecloningsitesofpSEAP2-ControlVector 26

List of Tables

Table I. SignalingPathwaysRepresentedinthePathwayProfilingSystems 5

Table II. CulturePlateConversion 14

Table III. PathwayProfilingVectorInformation 21

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3286-1 � Version No. PR15835


I. Introduction

ThePathway Profiling Systemsallowyoutoquicklyprofiletheeffectsofagivenstimulus,drugcandidate,orgeneofinterestonkeysignaltrans-ductionpathwaysin vivo.ThePathwayProfilingSystemscoveravarietyofsignaltransductionpathwaysineukaryoticcells.PathwayProfilingSys-temsareavailableforbroad-spectrumortargetedprofilingofkeysignalingpathways: • SEAPSystem—broadcoverage • SEAPSystem2—NuclearHormoneReceptors • LuciferaseSystem—broadcoverage • LuciferaseSystem2—PKC&Ca2+ • LuciferaseSystem3—Cellproliferation&differentiation • LuciferaseSystem4—Cellcycle • LuciferaseSystem5—Inflammation&CellproliferationEachkit iscomposedofseveraldifferentreportervectors thatcontainaspecificcis-actingDNAsequence(enhancerelement)andasensitivere-portergene.Thus,youcanmonitorthebindingoftranscriptionfactorstoenhancerelementsandscreenfortheinductionofkeysignalingpathways.TableIshowsthesignalingpathwaysrepresentedinthePathwayProfil-ingSystems.Foralistofvectorsetsprovidedineachsystem,seeListofComponents(SectionIV).Pathway profiling allows you to obtain preliminary evidence regardingtheroleofyourgeneordrugcandidateintheactivationofkeysignalingpathways.AlongwithaProfilingSystemvectorset,eachsystemincludesallthereagentsforperformingstandardcalciumphosphatetransfections.Figure1illustratesthegeneralschemeforperformingthepathwayprofilingprocedure.Assaymateri-

tablei.signalingpathwaysrepresentedinthepathwayprofilingsystems

cis-acting Transcription SignaltransductionEnhancerElement Abbrv. factor(s) pathway(s)

Activatorprotein1 AP1 c-jun/c-fos JNK cAMPresponseelement CRE ATF2/CREB JNK/p38&PKAE-boxDNAbindingelement E-box Myc/Max cellproliferationE2FDNAbindingelement E2F E2F/DP1 cellcycleprogressionEstrogenresponseelement ERE estrogenreceptor estrogenreceptorGlucocorticoidresponseelement GRE GR glucocorticoid/HSP90Heatshockresponseelement HSE HSF heatshockresponseIFN-γactivationsequence GAS STAT1/STAT1 proliferation/inflammationInterferon-stimulated ISRE STAT1/STAT2 proliferation/inflammationresponseelementNuclearfactorofactivatedT-cells NFAT NFAT PKC&Ca2+/calcineurinNuclearfactorofκBcells NFκB NFκB NFκBp53responseelement p53 p53 cellgrowth/apoptosisRetinoicacidresponseelement RARE RAR retinoicacidreceptorRbresponseelement Rb Rb cellcycleprogressionSTAT3responseelement STAT3 STAT3/STAT3 proliferation/inflammationSerumresponseelement SRE Elk-1/SRF MAPK/JNKThyroidresponseelement TRE Thyroidreceptor thyroidhormonereceptor



I. Introduction continued

alstodetectSEAPorluciferasearenotincluded;seeRelatedProducts(Sec-tionX)fororderinginformation.

SEAP and Luciferase—ideal reporters for studying signal transduction

ThePathwayProfilingSystemsoffertworeporters—SEAPandluciferase—soyoucanselectthedetectionmethodthatisbestsuitedforyourexperiments.Se-cretedalkalinephosphatase(SEAP)andluciferaseprovideseveraladvantagesforuseastranscriptionalreporters.Bothreporterproteinsaredetectableoverawidelinearrangemakingthemwellsuitedforcomparativeanalysis.Standardluciferaseassaysrequirelysisoftransfectedcells,whereasSEAPactivityisdetectedintheculturemedium—nocelllysisisrequired.BecauseSEAPissecretedintothemedium,youcancollectsamplesfromthesamecellcultureatvarioustimepoints(i.e.,time-coursestudies)withoutdisruptingthecells.Additionally,thesametransfectedcellscanbeuseddirectlyforfurtherinvestigationusingothermethods,suchasRNAorWesternblot-ting.Regardlessofthereporter,SEAPandluciferaseassaysareextremelysensitive,andbothreporterscanbedetectedusingaluminometer,liquidscintillationcounter,orx-rayfilm.WerecommendusingtheGreatEscAPe

Figure 1. General scheme for the Pathway Profiling procedure. Eachsystemiscomposedofseveral different vector constructs for screening key signal transduction pathways.Alongwitheachvectorset,sufficientreagentsareincludedforperforming50calciumphosphatetransfections.Reagentstodetectreportergeneactivityarenotincluded.

Reporter Gene (SEAP or luciferase)

Pathway Profiling Vectors

cis-acting Enhancer Element —

Transfect cell line with the appropriate vector in side-by-side cell cultures.

Incubate 2� hrsAdd stimulus

•

••

Assay & DetectReporter Activity



I. Introduction continued

ChemiluminescenceDetectionKit(Cat.Nos.631701,631702)withthePathwayProfilingSEAPSystem.Alternatively,theGreatEscAPeSEAPFluorescenceDetectionKit(Cat.No.631704)canalsobeused.WerecommendtheLu-ciferaseReporterAssayKit(#K2039-1)forusewiththePathwayProfilingluciferasereportervectors.

Onceyouhaveidentifiedthepathwayaffectedbyyourstimulus,useoneoftheTransFactorKitstostudythetranscriptionfactorstimulationin-depth;SeeRelatedProducts(SectionX)fororderinginformation.

Protocol No. PT3286-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR15835 �


II. Pathway Profiling Vectors

IneachPathwayProfilingvector,thespecificcis-actingDNAbindingsequenceislocatedupstreamfromoneoftwopromoters:theTATA-likepromoter(PTAL)regionfromtheherpessimplexvirusthymidinekinase(HSV-TK)promoterorjustitsTATAbox(PTA).Thesepromotersprovideoptimalinductionofthereporter,whileprovidingverylowbackground.Figure2showssomeex-amplesofstimulithattargetPathwayProfilingenhancerelements,therebyactivatingsignalingtransductionpathways.Additionalvectorinformation,suchascompletesequenceandvectormaps,canbedownloadedfromourwebsiteatvectors.clontech.com. Forrecommendeddiagnosticdigestsandgeneralizedillustrationsofthevectormaps,seetheAppendix.Control VectorsEach kit is supplied with a negative control vector to determine unin-ducedbackground levels of reportergeneactivity.The negative controls(pTAL-SEAP,pTAL-Luc,pTA-SEAPorpTA-Luc) lack theenhancerelement,but containapromoterand reportergene.Thevaluesobtainedwith thecontrol vectors can be subtracted from your experimental values. Ad-ditionally,youcanuse thesevectors tostudyyourownputativecis-act-ing enhancer element by cloning it into the MCS.The positive controlvector, pSEAP2-Control (provided in the original SEAP System, Cat. No.631910), is necessary for optimizing the SEAP assay. For more informa-tionregardingtheuseofthesecontrols,seeSectionVI.AortheAppendix.

Figure 2. An overview of the cis-acting enhancer element vector constructs in the Pathway Profiling Systems.Anexampleofastimulusthatwillactivatethebindingoftranscriptionfactorstoitsresponseelementisshowntotheleftofthefigure.ThePTALandPTApromotersprovideoptimalinductionofthereporter,whileprovidingverylowbackground.*InthecaseofRb,bindingoftheproteintotheresponseelementrepressesexpressionofthereportergene.

Response Element Promoter Reporter

AP1AP1(PMA)CREEREE2FGREHSEISREE-box (Myc)NFκBNFATRARERb*SRETRE

Stimulus

serumPMAforskolinestrogengrowth factorsglucocorticoids42C heat growth factorsserum/growth factorsTNF or IL-1PMA/Ca2+

retinoic acidRb proteinserumthyroid hormone

SEAP or luciferaseLuciferaseSEAP or luciferaseSEAPLuciferaseSEAP or luciferaseSEAP or luciferaseLuciferaseSEAPSEAP or luciferaseSEAP or luciferaseSEAPLuciferaseSEAP or luciferaseSEAP

Activation*

TALTATALTATALTALTALTALTALTAL, TATAL, TATATATALTA



III. Products for Signal Transduction Research

Clontechoffersafulllineofproductstofacilitateyoursignaltransductionresearch.InadditiontothePathwayProfilingSystems,severalcis-actingPathwayProfilingVectorsareavailableseparately;someareavailablewithdestabilizedenhancedgreenfluorescentprotein(dEGFP)asthereporter.Thefollowingisanoverviewofourcurrentproductline.Forthelatestprod-uctinformation,visitourwebsiteatwww.clontech.com,orseeSectionX(RelatedProducts)fororderinginformation.

• TransFactor Kits:Forrapiddetectionoftranscriptionfactoractivitiesincytosolicandnuclearextracts,theTransFactorKitsuseanenzyme-linkedimmunosorbentassay(ELISA)-basedformat.Thismethodiseasier,safer,andmoresensitivethantraditional(EMSA).Kitscomeintwoformats:thepathway-targetedformatallowsyoutoinvestigateonetranscriptionfactorresponseindepth,andthePathway-Profilingformatallowsyoutoinvestigatethefactor(s)inducedinreposetoaninflammationreaction.

• In Vivo Kinase Assay Kits:Afteryou’veidentifiedwhichpathwayyourgenehasactivated,youcanuseanyoftheIn VivoKinaseAssayKitstodeterminewhetheryourgeneordrugcandidateofinterestinducesanincreaseinkinaseactivityinaspecificsignalingpathway.Unlikethecis-actingenhancerelementsinthePathwayProfilingvectors,theIn Vivo Kinasevectorscontainatranscriptionalactivator(transactiva-tor)thatisspecificforonekeysignalingpathway.KitsareavailableforstudyingtheMAPK,p38K,JNK,andPKApathways.

• Kinase Expression Vector Set: Thisvectorsetconsistsofthreevectors,eachconstitutivelyexpressingoneofthefollowingkinases—MEK1,MEKK1,andPKA.TheseproteinkinasesareidealpositivecontrolsfortheIn VivoKinaseAssayKits,orforanyexperimentthatrequiresexpressionofthesekinases.

• EGFP and DsRed Signaling Probes:Tovisualizetheeffectsofageneorstimulusonlivingcells in vivo,weofferavarietyofEGFPandDsRedSignalingProbes.Wedesignedmammalianexpressionvectorswhichconstitutivelyexpressafusionofenhancedgreenfluorescentproteinorredflorescentproteintoakeysignaltransductionmolecule.Oncetransfected,youcanstudysignaltransductionasithappensinrealtime.

• Dominant Negative Vector Sets:Forstudyingavarietyofpathways,we offer the Dominant NegativeVector Sets, which constitutivelyexpresshighlevelsofawild-typesignaltransductionmoleculeoritsdominant-negativemutant.Thesevectorsallowyoutolinkyourtargetgenetoaparticularpathwayorbiologicalprocess.Currently,vectorsetsareavailableforIκBα,CREB,p53,Raf,andRas.Forad-ditionalexperimentaloptions,RafandRassetsalsoincludevectorsthatexpressconstitutivelyactivevariants.

Protocol No. PT3286-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR15835 �


IV. List of Components

AllvectorsandHEPES-BufferedSaline(HBS)shouldbestoredat–20°C.Allothercomponentsshouldbestoredat4°C.

ThePathwayProfilingSystemscontainsufficientreagentsforapproximately50calciumphosphatetransfectionsin35-mmtissuecultureplates.

For broad-coverage spectrum profiling:Pathway Profiling SEAP System (Cat.No.631910)• 20 µg pTAL-SEAPVector(0.5µg/µl;negativecontrol)• 20 µg pSEAP2-ControlVector(0.5µg/µl;positivecontrol)

• 20 µg pAP1-SEAPVector(0.5µg/µl)

• 20 µg pCRE-SEAPVector(0.5µg/µl)

• 20 µg pGRE-SEAPVector(0.5µg/µl)

• 20 µg pHSE-SEAPVector(0.5µg/µl)

• 20 µg pNFAT-SEAPVector(0.5µg/µl)

• 20 µg pNFκB-SEAPVector(0.5µg/µl)

• 20 µg pMyc-SEAPVector(0.5µg/µl)

• 20 µg pSRE-SEAPVector(0.5µg/µl)

• 1 ml 2MCalciumSolution

• 7 ml 2XHEPES-BufferedSaline(HBS)

• 7 ml SterileH2O

Pathway Profiling Luciferase System(Cat.No.631911)• 20 µg pTAL-LucVector(0.5µg/µl;negativecontrol)• 20 µg pAP1-LucVector(0.5µg/µl)

• 20 µg pCRE-LucVector(0.5µg/µl)

• 20 µg pGRE-LucVector(0.5µg/µl)

• 20 µg pHSE-LucVector(0.5µg/µl)

• 20 µg pNFκB-LucVector(0.5µg/µl)

• 20 µg pSRE-LucVector(0.5µg/µl)



• 7 ml SterileH2O



IV. List of Components continued

To profile nuclear hormone receptor signaling pathways:Pathway Profiling SEAP System 2 (Cat.No.631920)• 20µg pTA-SEAPVector(0.5µg/µl)• 20 µg pERE-TA-SEAPVector(0.5µg/µl)• 20 µg pTRE-TA-SEAPVector(0.5µg/µl)• 20 µg pRARE-TA-SEAPVector(0.5µg/µl)• 1 ml 2MCalciumSolution


• 7 ml SterileH2O

To profile PKC & Ca2+ signal transduction pathways:Pathway Profiling Luciferase System 2 (Cat.No.631912)• 20 µg pTA-LucVector(0.5µg/µl;negativecontrol)• 20 µg pAP1(PMA)-TA-LucVector(0.5µg/µl)

• 20 µg pNFAT-TA-LucVector(0.5µg/µl)

• 20 µg pNFκB-TA-LucVector(0.5µg/µl)



• 7 ml SterileH2O

To profile cell proliferation & differentiation signaling pathways:Pathway Profiling Luciferase System 3(Cat.No.631913)• 20 µg pTAL-LucVector(0.5µg/µl;negativecontrol)• 20 µg pAP1-LucVector(0.5µg/µl)

• 20 µg pCRE-LucVector(0.5µg/µl)

• 20 µg pISRE-LucVector(0.5µg/µl)

• 20 µg pE2F-LucVector(0.5µg/µl)

• 20 µg pSRE-LucVector(0.5µg/µl)



• 7 ml SterileH2O



IV. List of Components continued

To profile cell cycle signaling pathways:Pathway Profiling Luciferase System 4(Cat.No.631914)• 20 µg pTA-LucVector(0.5µg/µl;negativecontrol)• 20 µg pE2F-TA-LucVector(0.5µg/µl)

• 20 µg pMyc-TA-LucVector(0.5µg/µl)

• 20 µg pp53-TA-LucVector(0.5µg/µl)

• 20 µg pRb-TA-LucVector(0.5µg/µl)



• 7 ml SterileH2O

To profile inflammation & cell proliferation pathways:Pathway Profiling Luciferase System 5(Cat.No.631915)• 25 µg pTA-LucVector(0.5µg/µl;negativecontrol)• 25 µg pSTAT3-TA-LucVector(0.5µg/µl)

• 25 µg pGAS-TA-LucVector(0.5µg/µl)

• 25 µg pISRE-TA-LucVector(0.5µg/µl)



• 7 ml SterileH2O



V. Additional Materials Required

• SEAP reporter gene assay(WerecommendourGreatEscAPeSEAPDetectionSystems,Cat.Nos.631701,631702and631704)

• Luciferase reporter gene assay (WerecommendourLuciferaseReporterAssayKit,#K2039-1)

• Cell culture plates or flasks• Tubes(12x75-mmsteriletubes)• Cell culture medium(appropriategrowthmediumformammaliancells

inculture)• Fetal bovine serum, newborn calf serum, or equivalent(tosupplement

thegrowthmedium)• Phosphate buffered saline(PBS;pH7.4) Finalconc. Toprepare2Lofsolution Na2HPO4 58 mM 16.5 g NaH2PO4 17 mM 4.1 g NaCl 68 mM 8.0 g

Dissolvetheabovecomponentsin1.8LofdeionizedH2O.AdjusttopH7.4with0.1NNaOH.AdddeionizedH2Otofinalvolumeof2L.Storeatroomtemperature.

• 1X Trypsin/EDTA(LifeTechnologies#25300-054)• 0.5-ml microcentrifuge tubes or 96-well flat-bottom microtiter plate

Chemiluminescenceassaysaregenerallyperformedin0.5-mlmicro-centrifugetubes.Alternatively,reactionscanbeperformedinawhiteopaque96-wellflat-bottommicrotiterplate,suchasthosefromXeno-poreorCostar.

• Luminometer(tubeorplate)or x-ray film Chemiluminescence detection of SEAP or luciferase activity can be

performedeitherwithaluminometer(tubeorplate)orviaexposureofx-rayfilmtoreactionsperformedinawhiteopaque96-wellflat-bottommicrotiterplate,suchasthosefromXenoporeorCostar.



VI. General Considerations

PLEASE READ THROUGH ENTIRE PROTOCOL BEFORE BEGINNING

A. Use of Controls Alwaysperformcontrolexperimentswiththeappropriatevectorsto

determinethebasallevelofSEAPorluciferaseactivityandtoensurethat the assay is optimized.The Pathway Profiling vectors may betransfectedintomammaliancellsbyavarietyoftechniques.However,amethodthatworkswellforonetypeofculturedcellmaybeinferiorforanother.Whenworkingwithacelllineforthefirsttime,comparethe transfection efficiencies of several transfection protocols usinga control vector expressing a reporter gene (e.g., pSEAP2-Control,pCMV-β,orpEGFP-C1or -N1Vector).We recommend the followingexperimentalprocedureswhenusingthissystem:

1.Negativecontrols Performinganegativecontrolisnecessarytodeterminebackground

signalsassociatedwiththeculturemediumandreporteractivity.Thiscanbedeterminedbytransfectingcellswiththeappropriatecontrol:pTAL-SEAP/LucorpTA-SEAP/Luc.Thevaluesobtainedfromsuchcontrolsshouldbesubtractedfromexperimentalresults.

2.Positivecontrolfortransfection—SEAPsystem(Cat.No.631910)only Performing a positive control is useful to confirm transfection

andtoverifythepresenceofactiveSEAPintheculturemedium.ExpressionandsecretionoffunctionalSEAPintransfectedcellscanbeconfirmedbyassaying25µlofculturemediumfromcellstransfectedwithpSEAP2-Control.Cellstransfectedwiththisplas-midshouldexhibithighSEAPactivitywithin24–72hoursaftertransfection.

B. Transfection Considerations 1.Performalltransfectionsintriplicate Eachconstructshouldbetransfected(andsubsequentlyassayed)

intriplicatetominimizevariabilityamongtreatmentgroups.Trans-fectionefficiencyistheprimarysourceofthisvariability.

2.Normalizingtransfectionefficiencies It is critical to include an internal control that will distinguish

differencesintranscriptionalinductionfromvariabilityintheef-ficiencyoftransfection(Sambrooket al., 1989).Thisiseasilydonebycotransfectingasecondplasmidthatconstitutivelyexpressesareportergene,whichcanbeclearlydifferentiatedfromSEAPandluciferase.ThelevelofexpressionfromthisgenecanthenbeusedtonormalizethelevelsofSEAPorluciferaseactivityamongdiffer-enttreatmentgroups.Reporterproteinsfrequentlyusedforthispurposeincludeβ-galactosidaseandEGFP,orSEAPforluciferaseandluciferaseforSEAP.

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3286-1 1� Version No. PR15835


VI. General Considerations continued

C. Optimization of Transfection

Theefficiencyofamammaliancelltransfectionisprimarilydependentonthehostcelllineused.Optimizationofthetransfectionparametersforeachcelltypeiscrucialtoobtainingconsistentlysuccessfultransfec-tions.Therefore,foreachcelltypeyouplantouse,performpreliminaryexperimentstodeterminetheoptimalcelldensity,amountandpurityofDNA,andtransfectionincubationtime.Forthepreliminaryexperi-ments,thehostcelllinecanbetransfectedwithareporterexpressionvector,suchaspSEAP2-ControlVector.Thesuccessofthetransfectioncanthenbeestimatedbyassayingforsecretedalkalinephosphatase.Oncethetransfectionparametershavebeenoptimized,theyshouldbekeptconsistentfromoneexperimenttothenexttoobtainreproducibleresults.

Thefollowing isageneralguidelineforoptimizingthetransfectionparameters.Itisbesttoperformaseriesofsmall-scaletransfections.Thiscanbedoneconvenientlyin12-wellor6-wellplates.Tooptimizecelldensity,keepingallotherparametersconstant,platehostcellsinindividualwellsofa6-wellplateatvaryingdensities(e.g.,5x104,1x105,2x105,4x105,and8x105).24–72hrspost-transfection,assayforreportergeneactivity.Recordresults.Repeattheexperimentonceortwicetoaccountforday-to-dayvariation.Choosethedensitywiththehighestreportergeneactivity.

Otherparameterscanbeoptimizedinmuchthesameway.Holdallothervariablesconstantwhilevaryingtheparameteryouaretesting.Transfectionincubationsshouldbemaximalat2–16hrsusingacal-ciumphosphatetransfectionprotocol.Youmaywanttotryincubationtimesfrom1–18hrsforoptimization.Aftertransfectionshavebeenoptimized,scaleuporscaledownasnecessaryforthesizeofcultureplateyouareusing(seeTableIIforcultureplateconversions).

tableii.cultureplateconversion

Size of Plate Growth Area Relative Area* Recommended (cm2) Volume 96well 0.32 0.04X 200µl24well 1.88 0.25X 500µl12well 3.83 0.50X 1.0ml 6well 9.4 1.20X 2.0ml 35 mm 8.0 1.00 X 2.0 ml60mm 21 2.60X 5.0ml 10cm 55 7.00X 10.0mlFlasks 25 3.00X 5.0ml 75 9.00X 12.0ml*Relativeareaisexpressedasafactorofthegrowthareaofa35-mmcultureplate.



VII. Pathway Profiling System Procedure

Before you Start:

• IMPORTANT: Aftertransfection,itisimportanttoremoveserumfromthemediumtoensureproperinductionofthereporter.Seruminducesvarioussignalingpathwayscausinghighbackground.

• Althoughyoumayuseanyprotocoldesignedfortransfectingmammaliancultures,thePathwayProfilingsystemcontainsthenecessaryreagentstoperformastandardcalciumphosphatetransfectionprocedure.

• Designamethod for studyingadrugcandidateorgeneof interest.Forexample,16–24hrsafter transfection,addastimulus to thecul-ture medium containing 0–0.5% serum.To determine the maximumresponsegivenbyyourstimulus,performatime-coursestudybycol-lectingsamplesatvarioustimepoints.FortheSEAPSystems,youcancollectsamplesfromasinglecellculture;forluciferasesystems,youmustsetupmultiplecellculturestocollectatdifferenttimepoints.Theparametersofthetime-coursemustbedeterminedempiricallyforeachexperiment.

Alternatively,tostudytheeffectsofageneofinterest,cotransfecteachprofilingvectorwithanexpressionvectorcontainingyourgene.Typi-callyineachwellofa6-wellplate,use1µgofprofilingvectorwithyourexpressionvector.Foroptimalinductionofthereporterbyyourgeneofinterest,werecommendsettinguptransfectionswithdifferentamountsofexpressionplasmidcontainingyourgene.Theseamountsmustbedeterminedempiricallyforeachexperiment.Tostudytheeffectsofadrugcandidate(s)andyourgeneofinterest,addyourstimulustothetransfectedcells(asdescribedabove)andassayforreporteractivity.

• BesuretoincludecontrolsasdescribedinSectionVI.A.



VII. Pathway Profiling System Procedure continued

Thefollowingprotocolisdesignedforusewithadherentculturesgrowingin 35-mm tissue-culture plates usingastandardcalciumphosphatetransfectionprotocol.Ifyouareusingplates,wells,orflasksofadifferentsize,adjustthecomponentsinproportiontothesurfaceareaofyourcontainer.SectionVI.Ccontainshelpful information foroptimizing transfectionproceduresandconvenientinformationforcultureplateconversions.

All steps of the following protocol should be performed in a sterile tissue culture hood.

1.Platethecellsthedaybeforethetransfectionexperiment.Thecellsshouldbe50–80%confluentthedayoftransfection.Generally,weplate4x105cells/35-mmplate.

2.0.5–3hrspriortotransfection,replaceculturemediumonplatestobetransfectedwith2mloffreshculturemediumper35-mmplate.

3.Foreachtransfection,prepareSolutionAandSolutionBinseparatesteriletubes.

To reduce variability when transfecting multiple plates with the same plasmid DNA, prepare a master mix with enough of Solu-tions A and B for each transfection:

Solution A: add components in the following order: ~1µg PlasmidDNA SterileH2O 12.4µl 2MCalciumSolution 100µl Totalvolume

Solution B: 100µl2XHBS 4.CarefullyandslowlyvortexSolutionBwhileaddingSolutionA,

dropwise.(Alternatively,blowbubblesintoSolutionBwitha1-mlsterilepipetteandanautopipettorwhileaddingSolutionAdrop-wise.)

5.Incubatethetransfectionsolutionatroomtemperaturefor20mins. 6.Gentlyvortexthetransfectionsolution,andthenaddthesolution

dropwisetocultureplatemedium.Add200µloftransfectionsolu-tionper35-mmplate.

7.Gentlymoveplatesbackandforthtodistributetransfectionsolutionevenly.Donotrotateplatesasthiswillconcentratetransfectionprecipitateinthecenterofthewellorplate.

8.Incubateplatesat37°Cfor2–12hrsinaCO2incubator. 9.Removecalciumphosphate-containingmediumandwashcells

withmediumor1XPBS.

Protocol No. PT3286-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR15835 1�


VII. Pathway Profiling System Procedure continued

A. Low Transfection Efficiency • Poorprecipitateformation Solution: Addition of the calcium/DNA (Solution A) to the

2X HBS (Solution B) should be performed dropwise and withcontinuousmixing.AddingSolutionAtooquicklyorwithtoolittlemixingcanresultinapoorprecipitate.

• PoorqualityDNA Solution: TheA260/A280ratiooftheplasmidDNAshouldbe≥1.7. • pHnotoptimal Solution: The pH of the HBS should be between 7.05 and 7.12.

However,duringprolongedstorage,thepHofthesolutionmaychange;therefore,usethePathwayProfilingSystemwithintheshelflife indicatedon theaccompanyingProductAnalysisCertificate(PAC).

B. Variable Transfection Efficiency in Experiments Therewillalwaysbesomevariabilityintransfectionefficiencies.Werec-

ommendperformingtransfectionsintriplicatetominimizethevariability.

• Variablecelldensity Solution: Keepcelldensityconstantafteroptimizingtransfection

procedures.Generallyweuseculturesthatare50–80%confluentatthetimeoftransfection.

•Suboptimalcellgrowth Solution: Keepcellshealthyinculture.Cellsshouldbeinmid-log

phasegrowthwhenplatedfortransfection.Transfectionefficien-ciesmaydecreaseforcelllinesthathavebeenpassagedfortoomanygenerations.

VIII. Troubleshooting Guide

10.Feedplatewith2mloffreshmediumcontaininglowserum(0–0.5%)andincubateat37°Cuntilneededforassay(~16–24hrs).

Note: Aftertransfection,itisimportanttoremoveserumfromthemediumtoensureproper inductionof thereporter.Serumcaninducevarioussignalingpathwayscausinghighbackground.

11.Proceed with your experiment, then assay for the appropriatereportergene.



IX. References

Chen,C.&Okayama,H.(1988)Calciumphosphatemediatedgenetransfer:AhighlyefficienttransfectionsystemforstablytransformingcellswithplasmidDNA.BioTechniques6:632–638.

Cullen,B.R.&Malim,M.H.(1992)Secretedplacentalalkalinephosphataseasaeukaryoticreportergene. Meth. Enzymol.216:362–368.

Eggermont,J.&Proudfoot,N.(1993)Poly(A)signalsandtranscriptionalpausesitescombinetopreventinterferencebetweenRNApolymeraseIIpromoters.EMBO J. 12:2539–2548.

Freshney,I.R.(1993)Culture of Animal Cells,ThirdEdition(Wiley-Liss,NewYork,NY).

Kain,S.R.&Ganguly,S.(1995)OverviewofGeneticReporterSystems.InCurrent Protocols in Molecular Biology,Ed.Ausubel,F.M.et al.,(Wiley&Sons,NY)Unit9.6.

Kain,S.R.(1996)Useofsecretedalkalinephosphataseasareporterofgeneexpressioninmammaliancells.Methods in Molecular Biology,vol.63(HumanaPress,Totowa,NJ).

Sambrook,J.,Fritsch,E.F.&Maniatis,T.(1989)Molecular Cloning: A Laboratory Manual,ColdSpringHarborLaboratoryPress(ColdSpringHarbor,NY).



X. Related Products

ForthelatestandmostcompletelistingofallClontechproducts,pleasevisitwww.clontech.com

• CalPhos™MammalianTransfectionKit 631312• CLONfectin™ 631301• GreatEscAPe™SEAPChemiluminescenceDetectionKit 631701 631702• GreatEscAPe™SEAPFluorescenceDetectionKit 631704

In Vivo Kinase Assay Kits• KinaseExpressionvectorSet 631927

TransFactor and TransFactor Profiling Kits• NFκBKit 631916• ProfilingKit-Inflammation1 631919

Dominant Negative Vector Sets• p53 631922• CREB 631925• Ras 631924• Raf 631926• IκBα 631923



X. Related Products continued

Pathway Profiling Reporter and Control Vectors• pAP1(PMA)-TA-LucVector 631906• pAP1(PMA)-TA-SEAPVector 631907• pAP1-SEAPVector 631903• pSRE-SEAPVector 631901• pGRE-SEAPVector 631902• pNFκB-SEAPVector 631905• pNFκB-LucVector 631904• pCMV-βVector 631719

NucleoBond® Plasmid Kits• MidiKit 635929 635930 635931

• MaxiKit 635933 635934 635935



Appendix: Pathway Profiling System Vectors

tableiii.pathwayprofilingvectorinformation

Vectors* Size Restriction Fragment (kb) sites size(s) Negative ControlspTAL-SEAP 4.8 Xho I 4.8kbpTAL-Luc 5.0 HindIII,SphI,Xba I 3.3,1.0&0.7kbpTA-SEAP 4.7 BglII 4.7kb BglII&Not I 4.5&0.2kbpTA-Luc 4.9 BglII 4.9kb NheI,HindIII 4.8&0.1kbPositive ControlpSEAP2-Control 5.1 HindIII,Xba I, 3.6&1.5kb AseI&BamHI 2.0,1.8&1.3kbProfiling VectorspAP1-SEAP 4.9 NheI,HindIII,XbaI 3.1,1.6&0.2kbpAP1-Luc 5.0 HindIII&SphI 4.3&0.7kbpAP1(PMA)-TA-Luc 4.9 Bgl II 4.9kb XbaI 3.2&1.7kbpCRE-SEAP 4.9 HindIII,SacII,XbaI 3.1,0.9,0.6&0.3kbpCRE-Luc 5.0 HindIII&SphI 4.3&0.7kbpE2F-Luc 5.0 BglII 5.0kb HindIII,SphI 4.3&0.7kbpE2F-TA-Luc 4.9 NheI&XbaI 3.0&1.9kbpERE-TA-SEAP 4.7 BglII 4.7kb NotI&NheI 4.5&0.2kbpGAS-TA-Luc 4.9 BglII&XbaI 3.0&1.9kbpGRE-SEAP 4.9 BglII&XbaI 3.3&1.6kbpGRE-Luc 5.0 HindIII&SphI 4.3&0.7kbpHSE-SEAP 4.9 HindIII,SacII,XbaI 3.1,0.9,0.6&0.3kbpHSE-Luc 5.0 HindIII&SphI 4.3&0.7kbpISRE-Luc 5.0 BglII 5.0kb HindIII,SphI 4.3&0.7kbpISRE-TA-Luc 4.9 BglII&XbaI 3.0&1.9kbpMYC-SEAP 4.8 HindIII,SacII, XbaI 3.1,0.9,0.6&0.2kbpMYC-TA-Luc 4.8 NheI&XbaI 3.0&1.8kbpNFκB-SEAP 4.9 HindIII&Xba I 3.3&1.6kbpNFκB-TA-Luc 4.9 BglII 4.9kb NheI,HindIII 4.8&0.1kbpNFκB-Luc 5.0 HindIII&XbaI 3.3&1.7kbpNFAT-SEAP 4.9 HindIII,SacII, XbaI 3.1,0.9,0.6&0.3kbpNFAT-TA-Luc 4.9 BglII 4.9kb NheI,HindIII 4.7&0.2kbpp53-TA-Luc 4.9 KpnI&XbaI 3.0&1.9kb

The complete sequence information for the Pathway Profiling SystemVectors can be downloaded from our web site at vectors.clontech.com.TableIIIshowsthevectorsize,diagnosticrestrictionsites,andfragmentsizesforallthesevectors.



Appendix: Pathway Profiling System Vectors continued

tableiii.pathwayprofilingvectorinformation, continued

Vectors* Size Restriction Fragment (kb) sites size(s)

pRARE-TA-SEAP 4.7 BglII 4.7kb NotI&MluI 4.5&0.2kbpRb-TA-Luc 4.9 XhoI&XbaI 3.0&1.8kbpSRE-SEAP 4.9 HindIII 4.9kbpSRE-Luc 5.0 HindIII&SphI 4.3&0.7kbpSTAT3-TA-Luc 4.9 BglII& XbaI 3.0&1.9kbpTRE-TA-SEAP 4.7 BglII 4.7kb KpnI&NotI 4.5&0.2kb*TheidentityofeachPathwayProfilingvectorandenhancerelementwasconfirmedbysequencing.




Figure 3. Generalized map of the Pathway Profiling Vectors.PathwayProfilingvectorscontainoneoftwopromoters:theentireTATA-likepromoter(PTAL)regionfromthethymidinekinasebasalpromoteroftheherpessimplexvirus(HSV-TK)orjustitsTATAbox(PTA).ThepromoterislocatedupstreamoftheSEAPorluciferasecodingsequence.TheSEAPorluciferasecod-ingsequenceisfollowedbytheSV40latepolyadenylationsignaltoensureproper,efficientprocessingoftheSEAPorluciferasetranscriptineukaryoticcells.Asynthetictranscriptionblocker(TB)islocatedupstreamoftheresponseelementforreducingbackgroundtranscrip-tion(Eggermont,J.&Proudfoot,N.,1993).TableIIIshowsalistoftheresponseelementsandtheconsensussequencesinthePathwayProfilingSystem.Formoreinformationabouteachvector,visitourwebsiteatvectors.clontech.com.

Pathway Profiling Vector

pUCori

Ampr

SV40poly A

f1ori

SEAP/Luc

TB

TB=Transcription Blocker

▲▲ cis-acting Enhancer Element

AP1AP1 (PMA)

CREE2FEREGASGREHSEISRE

MYC (e-box)NFATNFκBRARE

RbSRE

STAT3TRE

Each vector has one of the following enhancer elements:P

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3286-1 2� Version No. PR15835



Figure 4. Map and multiple cloning sites of pTAL-SEAP/Luc Vectors.pTAL-SEAP/LucVectorscanbeusedtodeterminethebackgroundsignalsassociatedwiththeculturemedium.Ad-ditionally,thesevectorscanbeusedforstudyingputativeenhancerelements,whichcanbeclonedintotheMCS.pTAL-SEAP/LuccontainstheentireTATA-likepromoter(PTAL)regionfromthebasalpromoterof theherpessimplexvirus thymidinekinase (HSV-TK)promoter.PTALensuresoptimal inductionofthereporterwhileprovidingverylowbackground.TheSEAPorluciferasecodingsequenceisfollowedbytheSV40latepolyadenylationsignaltoensureproper,efficientprocessingoftheSEAPorluciferasetranscriptineukaryoticcells.Asynthetictranscriptionblocker(TB)islocatedupstreamoftheMCSforreducingbackgroundtranscrip-tion(Eggermont,J.&Proudfoot,N.,1993).

Kpn I Mlu I Nhe I Xho IGGTACCGAGCTCTTACGCGTGCTAGCCCGGGCTCGAGATCT

1•

20•

10•

30•

40•

Bgl II

MCS(1–41)

pTAL-SEAP/LucAmpr

SV40poly A

TB = Transcription blocker

TB

SEAP or Luc

pUC ori

f1ori

PTAL




Figure 5. Map and multiple cloning sites of pTA-SEAP/Luc Vector.pTA-SEAP/LucVectorcanbeusedtodeterminethebackgroundsignalsassociatedwiththeculturemedium.Additionally,thesevectorscanbeusedforstudyingputativeenhancerelements,whichcanbeclonedintotheMCS.pTA-SEAP/LuccontainsjustaTATAbox(PTA)thatensuresoptimalinductionofthereporterwhileprovidingverylowbackground.TheSEAPandluciferasecodingsequencesarefollowedbytheSV40latepolyadenylationsignaltoensureproper,efficientprocessingofthetranscriptineukaryoticcells.Asynthetictranscriptionblocker(TB)islocatedupstreamoftheMCSforreducingbackgroundtranscription(Eggermont,J.&Proudfoot,N.,1993).

Kpn I Mlu I Nhe I Xho IGGTACCGAGCTCTTACGCGTGCTAGCCCGGGCTCGAGATCT

1•

20•

10•

30•

40•

Bgl II

pTA-SEAP/Luc�.� kbAmpr

SV40poly A


TB

SEAP/luciferase

pUC ori

f1ori

PTA

MCS(1–41)




Figure 6. Map and multiple cloning sites of pSEAP2-Control Vector. Uniquerestrictionsitesareinbold.pSEAP2-ControlcontainstheSV40earlypromoterinsertedupstreamoftheSEAPgeneandtheSV40enhancerinserteddownstream.pSEAP2-ControlconstitutivelyexpressesSEAPinmostcelltypes,whichmakesitidealforestablishingtransfectionefficiencyandoptimizingyourSEAPassaydetectionmethod.Asynthetictranscriptionblocker(TB)islocatedupstreamoftheMCSforreducingbackgroundtranscription(Eggermont,J.&Proudfoot,N.,1993).

Note on effects of SV40 large T antigen (COS cells):ThespecificlevelofexpressionforthepSEAP2-ControlVectorislikelytovaryindifferentcelltypes.ThismaybeparticularlytrueforcelllinescontainingtheSV40largeTantigen,suchasCOScells.ThelargeTantigenpromotesreplicationoftheSV40origin,sequencesofwhicharefoundinthepromoterregionofthepSEAP2-ControlVector.ThecombinationofthelargeTantigenandSV40originleadstoahighercopynumberofthesevectorsinCOScells,whichinturnmayresultinincreasedexpressionoftheSEAP reportergenerelativetovectorslackingtheSV40origin.

MCS A(1–41)

Not I(4949)

pSEAP2-Control5.1 kbAmpr

SV40poly A

SV40 oriPSV40e


MCS B(245–264)

BamH I(2302)

Xba I(1794)

Ase I(3623)

BamH I(481)

TB

SEAP

SV40enhancerpUC

ori

f1ori

GGTACCGAGCTCTTACGCGTGCTAGCCCGGGCTCGAGATCT

AAGCTTCGAATCGCGAATTCGCCCACCATGCTG

Mlu I Nhe I Xho I Bgl IIAsp718 I Kpn I

Srf I

20•

30•

40•

10•

Hind IIIBstB I EcoR INru I

SEAP250•

260•

270•

PSV40e

MCS A

MCS B



Notes

pathway profiling system user manual - takara9].pdf · tems are available for broad-spectrum or...

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