knockout rnai systems user manual - dartmouth collegetonyz/retroq.pdf · vii. transfection of ......

Knockout™ RNAi SystemsUser Manual

PT3739-1 (PR631582)Published 23 March 2006

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3739-1 � Version No. PR63158�

Knockout™ RNAi Systems User Manual

Table of Contents

I. Introduction 4

II. List of Components 11

III. Additional Materials Required 14

IV. General Considerations 15

V. shRNA Oligonucleotide Design 18

A. SelectingTargetSequences 18

B. DesigningOligonucleotides 19

VI. Cloning into RNAi-Ready pSIREN Vectors 21

A. AnnealingtheOligonucleotides 21

B. LigatingdsOligonucleotideintoRNAi-ReadypSIREN 21

C. TransformingFusion-Blue™CompetentCellswithrecombinantpSIREN 22

VII. Transfection of Recombinant pSIREN Vectors 24

VIII. Viral Delivery of pSIREN Constructs 26

IX. Analysis of Results and Troubleshooting Guide 27

X. References 29

XI. Related Products 32

Appendix: Vector Information 34

Protocol No. PT3739-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR63158� 3


List of Figures

Figure1. MechanismofRNAinterference(RNAi) 5

Figure2. SmallhairpinRNAs(shRNAs)generatedusinganoligonucleotideDNAsequence 8

Figure3. OverviewoftheKnockoutRNAiSystemsprocedure 15

Figure4. shRNAoligonucleotidesequencedesign 20

Figure5. ProceduresprovidedforviraldeliveryofrecombinantpSIREN 26

Figure6. RestrictionMapandCloningSiteoftheRNAi-ReadypSIREN-RetroQRetroviralVector 34

Figure7. RestrictionMapandCloningSiteoftheRNAi-ReadypSIREN-ShuttleVector 35

Figure8. RestrictionMapandCloningSiteoftheRNAi-ReadypSIREN-DNRVector 36

List of Tables

TableI. DeliveryoptionsforKnockoutRNAivectorsandsystems 10

TableII. Examplesofpublishedtargetsequences 20

Table of Contents continued

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3739-1 � Version No. PR63158�


I. Introduction

A. SummaryThehumangenomeprojecthasgeneratedthesequencesofthousandsofgenes(Aaronsonet al.,1996;Hillieret al.,1996),allowingresearcherstofocusonthequestionofgenefunctioninbiology.Akeyapproachto determining gene function has been the targeted "knockout" ofspecificgenes;geneinactivationisaccomplishedbydisruptionofthetargetgene'scodingsequenceandthenintroducingthealteredgeneintoembryonicstemcells.Animalmodelscarryingheterozygousandhomozygousgeneknockoutsprovidetheabilitytodeterminewhetheraparticulargeneisessentialandwhatfunctionsareperturbedbyitsloss. However, the amount of time and labor required to generateanimalknockoutmodelsisquiteextensive.Achievingsuchmodelsinsomaticcelllineshasalsoprovendifficult(Sedivy&Dutriaux,1999).Anothermethodforeliminatinggeneexpressiontakesadvantageofthephenomenonofpost-transcriptionalgenesilencing(PTGS).Specifi-cally,thecellularprocessofRNAinterference(RNAi)hasbeenusedtoeffectivelysilencegeneexpression(Figure1).RNAiisactivatedbyintroducingadouble-stranded(ds)RNAwhosesequenceishomologoustothetargetgenetranscript.TheexogenousRNAisdigestedinto21–23nucleotide(nt)smallinterferingRNAs(siRNAs),whichbindanucleasecomplextoformanRNA-inducedsilencingcomplex(RISC).TheRISCthen targets the endogenous gene transcripts by base-pairing andcleavesthemRNA(Hammondet al.,2001;Sharp,2001;Huntvagner&Zamore,2002;andNykanenet al.,2001).Incontrasttotraditionalknockout methods, specific gene silencing is achieved quickly andeasilyinbothanimalandcelllinemodels.TheKnockoutRNAiSystemsallowyoutoquicklyexpressfunctionalhairpinsiRNAmoleculesinmammaliancellsforthepurposeofsilenc-ingtargetgenes.ThesesystemsincludeRNAi-ReadypSIRENVectorsthat use the cell's own RNA Polymerase III (Pol III) to transcribe aspecificallydesignedsmallhairpinRNA(shRNA)usingthehumanU6promoter.ThehumanU6promoterprovidesahighlevelofexpressioninmanycelltypes(Kunkel&Pederson,1989),resultingintargetgenesuppression.FormapsanddetailedinformationontheRNAi-ReadypSIRENVec-tors,seetheAppendixortheVectorInformationPacket(s)providedwithyourproduct.

B. Study and mechanism of RNAiPTGSwasinitiallydescribedinplants,whenattemptstoaugmenttheexpressionofchalconesynthaseinpetuniasresultedintheeffectivesuppressionofboththeendogenousgeneaswellastheintroducedgene;thephenomenonwascoined"cosuppression"(Napoliet al.,1990).Later,theuseofantisenseRNA,asingle-stranded(ss)RNAsequence



Cleavage of dsRNA by Dicer

Initiatorstep

Effectorstep

Active RISC associateswith target transcript

dsRNA

siRNAs

Unwinding of siRNA

mRNA

siRNAs bind to nuclease complex

Cleavage of target transcript

Inactive RNA-induced Silencing Complexes (RISCs)

Active RISCs

mRNA

mRNA

mRNA

mRNA

mRNA

Figure 1. Mechanism of RNA interference (RNAi).

I. Introduction continued

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3739-1 6 Version No. PR63158�


thatiscomplementarytoaparticularmRNA,tosuppresstheexpres-sionofthepar-1geneinC. elegansyieldedintriguingresults:injectingsensestrandRNA(essentiallyduplicatingthepar-1genetranscript)alsoresultedinsilencingexpression(Guo&Kempheus,1995).ItwasfinallydeterminedthatdsRNAwasevenmoreeffectivethanssRNAingenesilencing;thephenomenonwascalled"RNAinterference"(Fireet al.,1998).ThecurrentmodeloftheRNAimechanismincludesbothinitiatorandeffectorsteps(forreviewsseeHutvagner&Zamore,2002;Hammondet al.,2001;andSharp,2001).TheinitiatorstepinvolvesthedigestionoftheinputdsRNAintosiRNAs21–23ntinlength.ThesesiRNAsareproducedbytheactionofanenzymeknownasDicer,whichbelongstotheRNAseIIIfamilyofdsRNA-specificribonucleasesandisevolu-tionarilyconservedinworms,plants,fungi,andmammals(Bernsteinet al.,2001).ThecleavageofinputdsRNAbyDicerisaccomplishedinaprocessive,ATP-dependentmanner,eventuallygenerating19–21bpsiRNAduplexeswitha3'overhangof2nt(Figure1).TheeffectorstepoccurswhenthesesiRNAduplexesbindtoanucleasecomplexandformtheRISC(Figure1).RISCisactivatedbytheATP-dependentunwindingofthesiRNAduplex.ActiveRISCthentargetsthenative,homologoustranscriptbybasepairingandsubsequentlycleavesthemRNAatapproximately12ntfromthe3'endofthesiRNA(Hammondet al.,2001;Sharp,2001;Huntvagner&Zamore,2002;andNykanenet al.,2001).AnamplificationstephasalsobeenproposedtoexplainthepotencyoftheRNAiprocess;theexogenousRNAiscopiedmanytimeseitherbeforeorafterthegenerationofthesiRNAs(Ham-mondet al.,2001;Sharp,2001;andHuntvagner&Zamore,2002).RNAi,then,canserveasapowerfultoolinthefieldoffunctionalge-nomics.BysimplydesigningandintroducingadsRNAsequencethatiscomplementarytoaregionofatargetgenetranscript,loss-of-functionphenotypescanbegeneratedquicklyandeasily.

C. Establishing RNAi in mammalian cellsRNAiasatechniqueforgenefunctionanalysishasbeenwell-establishedinplants,nematodes,protozoa,andDrosophila.Inparticular,studiesinC. elegansandDrosophila haveshownthatusingdsRNAtoinduceRNAiiseffectiveingeneratingmodelsfortheanalysisofgenesinvolvedincelldivisionordevelopment(Schumacheret al.,1998;Gonczyet al.,1999;Mooreet al.,1999;Jantsch-Plunger&Glotzer,1999;Chaseet al.,2000;Kennerdell&Carthew,1998).Inmammaliancells,establishingRNAiiscomplicatedbynonspecificgenesilencing.TransfectinglongdsRNAs(>30nt)activatesanantiviralresponse,whichcanbemediatedbyeitherproteinkinaseR(PKR)or




RNase L. PKR activity leads to a global repression of translation(Mancheet al.,1992),whileRNaseLcatalyzesnonspecificRNAdeg-radation(Minkset al.,1979).RNAiwasfinallyachievedinmammaliancellsbytheintroductionofsiRNAs(<30nt),mimickingtheinitiatorstepoftheRNAimechanism(Elbashiret al.,2001).TheintroductionofsiRNAsbypassestheantiviralresponseinmammaliancells,allowingfunctionalgenesuppression.Thus,siRNAscanbeusedtoestablishRNAiinmammaliancellsandtostudyspecificgenefunction.AvailabletechnologiesforgeneratingsiRNAsincludechemicalsynthesisandin vitrotranscription.EarlyexperimentsinmammaliancellsusedchemicallysynthesizedsiRNAs,whichwasaneffectivebutexpensivemeanstoinduceRNAi.Toreducethecost,amethodforgeneratingsiRNAusingin vitrotranscriptionwasemployedtoproducemultipledifferentsiRNAsthatcouldbeintroducedintocellsusingtransienttransfection;however,RNAiachievedinthiswayeventuallydiminishes.Anumberofgroupshavedesignedplasmidexpressionvectorstogener-atesustainedproductionofsiRNAsbytransientorstabletransfection.SomeofthesevectorshavebeenengineeredtoexpresssmallhairpinRNAs(shRNAs),whichareprocessedin vivointosiRNA-likemoleculescapableofcarryingoutgene-specificsilencing(Brummelkampet al.,2002;Paddisonet al.,2002;Paulet al.,2002;andYuet al.,2002).Afterconstructioniscomplete,thesevectorscontainaDNAsequencethatencodestheshRNAclonedbetweenaPolIIIpromoterandatranscrip-tionterminationsitecomprising4–5thymidineresidues.Thetranscriptisterminatedatposition2oftheterminationsiteandthenfoldsintoastem-loopstructurewith3'UU-overhangs(Figure2).TheendsoftheshRNAsareprocessedin vivo,convertingtheshRNAinto~21ntsiRNA-likemolecules,whichinturninitiateRNAi(Brummelkampet al.,2002).ThesevectorsrepresentadefiniteimprovementininitiatingRNAiincells;howevernotallcelltypesareeasytotransfectusingthesevectors.

D. Knockout™ RNAi SystemsClontechhasaddressedshRNAdeliveryproblemsbyofferingexpres-sionvectorsthatallowefficient,cost-effectiveshRNAdeliverytomanycelltypes.TheRNAi-Ready pSIREN-RetroQ Vector,RNAi-Ready pSIREN-RetroQ-ZsGreen Vector,andRNAi-Ready pSIREN-RetroQ-DsRed-Express Vector areretroviralshRNAexpressionvectors.Thevectorsareprovidedlin-earizedandreadyforligationwithadsDNAoligonucleotideencodinganshRNA.pSIREN-RetroQcontainsapuromycinresistancegenefortheselectionofstabletransfectants.Forthedirectdetectionofcellscontainingyourgenesilencingconstruct,pSIREN-RetroQ-ZsGreenand




pSIREN-RetroQ-DsRed-ExpressconstitutivelyexpresstheZsGreenandtheDsRed-Expressfluorescentprotein,respectively(Matzet al.,1999).Eachvectorisself-inactivatingandoptimizedtoeliminatepromoterinterferencefromtheupstreamLTRintheintegratedprovirus(July2002Clontechniques,Juliuset al.,2000).TheCMVPromoterinthe5'LTRproduceshighviraltitersinstandardHEK293-basedpackagingcelllinesby48hraftertransfection.SeetheAppendixortheprovidedVector Information Packet for the vector map.The Retroviral GeneTransferandExpressionUserManual(PT3132-1)providesprotocolsforpackagingrecombinantpSIREN-RetroQ,pSIREN-RetroQ-ZsGreen,orpSIREN-RetroQ-DsRed-Expressintoinfectious,replication-incom-petent particles.You then simply apply virus-containing media todividingtargetcells.RetroviralinfectionallowsyoutointroduceyourshRNAintovirtuallyanymitoticallyactivecellwithhighefficiencyandmoderateshRNAexpression.

Figure 2. Small hairpin RNAs (shRNAs) generated using an oligonucleotide DNA sequence.Thisexampleshowsatargetsequencederivedfromthecodingregionoftheβ-actingene(Harborthet al.,2001).ThistargetsequenceiscloneddownstreamofaPolIIIpromoterinanexpressionvectorforgenesilencinginmammaliancells.Ahairpinloopsequenceislocatedbetweenthesenseandantisensesequencesoneachcomplementarystrand.TheshRNAbehavesasansiRNA-likemoleculecapableofcarryingoutgene-specificsilencing.ThismechanismisemployedbythepSIRENvectorfamily.


3'-CACTTCTAGTTCTAGTAACGAAGTTCTCTCGTTACTAGAACTAGAAGTGAAAAAA-5'

5'-GTGAAGATCAAGATCATTGCTTCAAGAGAGCAATGATCTTGATCTTCACT T T T T T-3'

Hairpin loop

Target sense sequence

Target sense sequence

Target antisense sequence

Target antisense sequence

Terminator

5'-GUGAAGAUCAAGAUCAUUGCUUCAAGAGAGCAAUGAUCUUGAUCUUCACUU-3'

Transcription oftarget

Folding of shRNAtranscript throughcis-base pairing

shRNA

shRNA transcript

Target sequence (ds DNA)




TheRNAi-Ready pSIREN-Shuttle VectorisaplasmidshRNAexpressionvector that isalso linearizedandreadyfor ligation.pSIREN-Shuttlecanbeuseddirectly intransienttransfectionexperiments.pSIREN-Shuttlecanalsobeusedwith theAdeno-X™ExpressionSystem1(Cat.No.631513)tointroducetheshRNAsequenceintoanadenoviralgenome.StandardligationtechniquesareusedtotransfertheshRNAexpressioncassette(SEC)frompSIREN-Shuttletoareplication-defi-cient,Ad5genome(Mizuguchi&Kay,1999).SeetheAppendixortheprovidedVectorInformationPacketforthevectormap.TheAdeno-X™ExpressionSystem1UserManual(PT3414-1,includedwiththevec-tor)providesprotocolsfortransferringtheSECfrompSIREN-Shuttleintotheadenoviralgenome,producingrecombinantadenovirus,andinfectingtargetcells.AdenoviralgenetransferiseffectiveatinfectingbothdividingandnondividingmammaliancellsfortransientandhighshRNAexpression.The Knockout™ Adenoviral RNAi System 1isacompletekitforcon-structingrecombinantgenesilencingadenovirus.ThekitisbasedontheAdeno-XExpressionSystem1andusesaroutinein vitroligationmethodtotransferafunctionalSECfrompSIREN-Shuttle(includedin the kit) into adenoviral DNA.We also provide the Knockout™ Adenoviral RNAi System 2,whichisacompletekitforconstructingrecombinant shRNA-expressingadenovirususingmoreconvenientin vitroCre-loxPrecombination.TherecombinationreactiontransfersafunctionalSECfromaDonorVector(pSIREN-DNR)toanadenoviralAcceptorVector:pLP-Adeno-X-PRLSViralDNA,a∆E1/∆E3Ad5genome.TheRNAi-Ready pSIREN-DNR-DsRed-Express Vectorisalsoavailableseparately for use with theAdenoviral RNAi System 2 or with theAdeno-X™PromoterlessExpressionSystem2(Cat.No.631525).pSI-REN-DNR-DsRed-Express constitutivelyexpressestheDsRed-Expressfluorescentproteinfordirectlymonitoringthedeliveryefficiencyofyourgenesilencingconstructusingeitherfluorescencemicroscopyorflowcytometry(Matzet al.,1999).ThepSIREN Control Vector Set includes threepairsofvectors thatactaspositiveandnegativecontrolsforgenesilencingofthefireflyluciferase gene (GenBankAccession No. M15077).The vectors areprovidedreadyforuse.SeeTableIforaguidetochoosingtheappropriatevectororsystemforyourneeds.ThisUserManualprovidesprotocolsforgeneratingyourownshRNAconstruct.ProtocolsinthisUserManualincludeshRNAoligonucle-otide sequence design, annealing of shRNA oligonucleotides, liga-tionofannealedoligonucleotidesintoRNAi-ReadypSIRENvectors,andtransformationandtransfectionofpSIRENconstructs.Protocolsfor recombinant pSIREN viral packaging, production, and infection




TABLE I. DELIVERy OPTIONS FOR KNOCKOuT™ RNAi VECTORS AND SySTEMSa

Vector/System Delivery option(s)

RNAi-ReadypSIREN-Shuttle Transienttransfectionb

RNAi-ReadypSIREN-RetroQ Transient/stabletransfection RNAi-ReadypSIREN-RetroQ-ZsGreen andretroviralinfectionRNAi-ReadypSIREN-RetroQ-DsRed-Express

RNAi-ReadypSIREN-DNR-DsRed-Express Transienttransfectionc

KnockoutAdenoviral Transienttransfection RNAiSystem1(ligation-basedcloning) andadenoviralinfection

KnockoutAdenoviral Transienttransfection RNAiSystem2(Cre-loxPmediatedcloning) andadenoviralinfectiona UsethistableasaguidefordecidingwhichKnockoutRNAivectororsystemtouse.b You can also transfer your functional shRNA expression cassette (SEC) from pSIREN-Shuttle into an

adenoviralgenomebypurchasing theAdeno-XSystem1Viral DNA(linear;Cat.No.631026)and theAdeno-XAccessoryKit(Cat.No.631027).

c YoucanalsotransferyourfunctionalSECfrompSIREN-DNR-DsRed-ExpressintoanadenoviralgenomebyusingtheAdeno-XPromoterlessExpressionSystem2(Cat.No.631525)ortheKnockoutAdenoviralRNAiSystem2.

aredescribedinthe secondaryUserManual(s)thataccompanyyourKnockoutRNAiSystem (seeSectionVIII).ForviraldeliveryofyourpSIRENconstructs,westronglyrecommendthatyouthoroughlyreadthe secondaryUserManual(s)thataccompanyyourKnockoutRNAiSystembeforebeginningtheprocedure.ThesecondaryUserManu-als include important background information, tips, and additionaltroubleshootingsuggestionsforusingaspecificviralsystem.



II. List of Components

Storeallcomponentsat–20°C.

Thefollowingreagentsaresuitablefor20ligationsintotheRNAi-ReadypSIRENVector. In addition, each KnockoutAdenoviral System providessufficientreagentstogenerate5recombinantadenoviruses.

Thegene-specificsequencesoftheLuciferaseshRNAAnnealedOligonucle-otide,theNegativeControlshRNAAnnealedOligonucleotide,andthepSIRENControlVectorSetderivefromGenBankAccessionNo.M15077

RNAi-Ready pSIREN-RetroQ Vector (Cat.No.631526)

• 1 µg RNAi-ReadypSIREN-RetroQVector(linearized,25ng/µl)

• 20 µl LuciferaseshRNAAnnealedOligonucleotide(0.5pmol/µl)

• 20 µl NegativeControlshRNAAnnealedOligonucleotide(0.5pmol/µl)

• pSIREN-RetroQVectorInformationPacket(PT3737-5)

• RetroviralGeneTransferandExpressionUserManual(PT3132-1)

RNAi-Ready pSIREN-RetroQ-ZsGreen Vector (Cat.No.632455)

• 1 µg RNAi-ReadypSIREN-RetroQ-ZsGreenVector(linearized,25ng/µl)



• pSIREN-RetroQ-ZsGreenVectorInformationPacket(PT3777-5)


RNAi-Ready pSIREN-RetroQ-DsRed-Express Vector (Cat.No.632487)

• 1 µg RNAi-ReadypSIREN-RetroQ-DsRed-ExpressVector(linearized,25ng/µl)



• pSIREN-RetroQ-DsRed-ExpressVectorInformationPacket(PT3830-5)

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3739-1 1� Version No. PR63158�



RNAi-Ready pSIREN-Shuttle Vector (Cat.No.631527)

• 1 µg RNAi-ReadypSIREN-ShuttleVector(linearized,25ng/µl)



• pSIREN-ShuttleVectorInformationPacket(PT3736-5)

• Adeno-X™ExpressionSystem1UserManual(PT3414-1)

RNAi-Ready pSIREN-DNR-DsRed-Express Vector (Cat.No.632457)

• 1 µg RNAi-ReadypSIREN-DNR-DsRed-ExpressVector(linearized,25ng/µl)



• pSIREN-DNR-DsRed-ExpressVectorInformationPacket(PT3778-5)

Knockout™ Adenoviral RNAi System 1 (Cat.No.631528)

• 1 µg RNAi-ReadypSIREN-ShuttleVector(linearized,25ng/µl)



• 20 µl Adeno-X™ViralDNA(PI-SceI/I-CeuIdigested;250ng/µl)

• 25 µl I-Ceu I(5units/µl)

• 100 µl PI-Sce I(1unit/µl)

• 250 µl 10XDoubleDigestionBuffer

• 100 µl 100XBSA

• 100 µl Adeno-X™ForwardPCRPrimer(100ng/µl)

• 100 µl Adeno-X™ReversePCRPrimer(100ng/µl)

• Adeno-X™PCRScreeningPrimerSetProtocol-at-a-Glance(PT3507-2)

II. List of Components continued



• pSIREN-ShuttleVectorInformationPacket(PT3736-5)


Knockout™ Adenoviral RNAi System 2 (Cat.No.631529)

• 40 µl RNAi-ReadypSIREN-DNRVector(linearized,25ng/µl)



• 5x18 µl Adeno-X™LPPromoterlessReactionMixa

• 5 µl CreRecombinase

• 100 µl Adeno-X™LPPromoterlessPrimerMixb

• pSIREN-DNRVectorInformationPacket(PT3738-5)


• Adeno-X™ExpressionSystems2UserManual(PT3674-1)a ContainspLP-Adeno-XplasmidDNA(theAcceptorVector),Crereactionbuffer,andbo-

vineserumalbumin(BSA).

b ContainsForwardPrimer1,ForwardPrimer2,andReversePrimer;20µMeach.PrimerSequencescanbefoundontheProductAnalysisCertificate.

pSIREN Control Vector Set (Cat.No.631627)

• 40 µl pSIREN-RetroQ-LucVector(50ng/µl)

• 40 µl pSIREN-RetroQ-NegVector(50ng/µl)

• 40 µl pSIREN-Shuttle-LucVector(50ng/µl)

• 40 µl pSIREN-Shuttle-NegVector(50ng/µl)

• 40 µl pSIREN-DNR-LucVector(50ng/µl)

• 40 µl pSIREN-DNR-NegVector(50ng/µl)

II. List of Components continued



III. Additional Materials Required

Thefollowingmaterialsarerequiredbutnotsupplied:

• Fusion-Blue™ Competent Cells(Cat.No.636700)

• CLONfectin™ Transfection Reagent(Cat.No.631301)or CalPhos™ Mammalian Transfection Kit(Cat.No.631312)

• Falcon™ 6-well cell culture plates (Cat.No.353046)

• Falcon™ 12 x 75-mm sterile polystyrene tubes (Cat.No.352003)

• NucleoSpin® Multi-8 Plus Plasmid Kit (Cat.No.635976)

• NucleoBond® Plasmid Maxi EF Kit(Cat.No.635953)

• Thermal cycler

• Bovine serum albumin (BSA),10mg/ml

• T4 DNA ligase (NewEnglandBiolabs,Cat.No.M0202S).10XT4DNALigaseBufferisprovidedwiththeenzyme.

• Luciferase expression vector.WerecommendthepGL2ControlVectorfromPromega(Cat.No.E1611).

• Cell culture medium(withandwithoutFetalBovineSerum)

• Phosphate buffered saline (PBS) (pH 7.4)

FinalConc. Toprepare2L Na2HPO4 58 mM 16.5 g NaH2PO4 17 mM 4.1 g NaCl 68 mM 8.0 g Dissolve components in 1.8 L of distilled H2O. Adjust to pH 7.4 with

0.1NNaOH.AddddH2Otofinalvolumeof2L.Autoclave.Storeatroomtemperature.

• Trypsin/EDTA(VWR/HycloneNo.16777-166)



IV. General Considerations

shRNA sequence design(Section V)

Anneal complementary shRNA oligonucleotides (<30 min)

(Section VI.A)

Ligate annealed oligonucleotides into pSIREN vector (~3 hr)

(Section VI.B)

Troubleshooting(Section IX)

Transform E. coli with pSIREN construct (2–3 days)

(Section VI.C )

Identify suppressingpSIREN constructs

Transfection of cell line with pSIREN construct (2 –3 days)

(Section VII)

Viral packaging of recombinant pSIREN DNA

(Section VIII)

Infect target cellswith recombinant

pSIREN virus

Knockout™RNAi SystemsUser Manual(PT3739-1)

Figure 3. Overview of the Knockout™ RNAi Systems procedure.

PLEASE READ ENTIRE PROTOCOL BEFORE STARTING.

Figure3showsanoverviewoftheproceduredescribedinthisUserManual,whichprovidesprotocolsforshRNAoligonucleotidesequencedesign,an-nealingofshRNAoligonucleotides,ligationofannealedoligonucleotidesintoRNAi-ReadypSIREN,andtransformationandtransfectionofpSIRENcon-structs.



ProtocolsforrecombinantpSIRENviralpackaging,production,andinfec-tionaredescribedinthe secondaryUserManual(s)thataccompanyyourKnockoutRNAiSystem(seeSectionVIII).Westronglyrecommendthatyouthoroughlyreadall UserManualsbeforebeginningtheprocedure.

shRNA Oligonucleotide Design(SectionV)• Thesuccessofyourexperimentdependsonchoosingtheproper

targetsequencewithinyourgeneofinterestandtheproperdesignoftheshRNAoligonucleotides.Inaddition,wehighlyrecommendthatyoutestmorethanoneshRNAsequenceforageneofinter-est.

• PAGEpurificationofyourdesignedoligonucleotidesensuresthatahigherpercentageoftheoligonucleotideswillbefull-lengthandincreasesthechanceofcloningacompleteandfunctionalinsert.WhenusingPAGE-purifiedoligonucleotides,wetypicallyachieve80–90%ofcloneswiththerightinsert.

• WhentestingyourpSIRENconstructforfunctionality,youwillneedagene-specificassaytotestforthesuppressionofGeneX.Examplesofgene-specificassaysthatcanbeusedinclude:

• WesternblotwithanantibodytoProteinX • RT-PCRusingGeneXprimers.Besureyoucandiscriminate

PCRproductsgeneratedfromgenomicDNAfromtrueRT-PCR products.

• NorthernblotwithGeneXprobe • FunctionalassayforProteinX

• ProLabelScreeningKits.OurscreeningkitsallowfastandquantititativechemiluminescentmeasurementofexpressionlevelsofanygenefusedtotheProLabel tag.Thekitsaresuppliedintwoformats,aCreatorformatforgenesalreadyclonedintotheCreatorbackbone(Cat.No.631542)andanIn-Fusion format for PCR cloning of precise, directionalconstructs(Cat.No.631724).Formoredetails,pleaseseetheProLabelScreeningKitUserManual(PT3789-1).

Transfection of Recombinant pSIREN Vectors(SectionVII)• ThetransfectionprotocolincludedinthisUserManualisintended

forthescreeningoffunctionalshRNAconstructsandgenesilenc-ingexperimentsusingtransfection.Forperforminggenesilencingexperimentsusingviralinfection,pleaserefertothesecondaryUserManualsincludedwithyourKnockoutRNAiSystem.ThesecondaryUserManualsdescribetheprotocolsforviralpackagingandinfec-tionusingpSIRENconstructs.

IV. General Considerations continued



• If a transfection method is already established for your cell linemodel,proceedwiththoseconditions.Itisimportanttokeepopti-mizedparametersconstanttoobtainreproducibleresults.

• ToensurethepurityoftheDNA,isolateallplasmidsfortransfectionusingaNucleoBondPlasmidMaxiEFKit(Cat.No.635953)orbyCsCldensitygradientpurification(Sambrook&Russell,2001).

IV. General Considerations continued



V. shRNA Oligonucleotide Design

Thissectiondescribestheprocessofidentifyingtargetsequenceswithina gene of interest and designing the corresponding oligonucleotides togeneratetheshRNA.

Whenpossible,avoidselectingtargetsenseorantisensesequencesthatcontainaconsecutiverunof3ormorethymidineresidues;apoly(T)tractwithinthesequencecanpotentiallycauseprematureterminationtheshRNAtranscript.

A. Selecting Target Sequences 1.Choosearegionof19nucleotides.Donotselectsequenceswithin

the5' and3'untranslated regions (UTRs)nor regionsnear thestartcodon(within75bases)asthesemaybericherinregulatoryproteinbindingsites(Elbashiret al.,2001).UTR-bindingproteinsand/ortranslationinitiationcomplexesmayinterferewithbindingoftheRISC.

2.CalculatetheGCcontentoftheselected19-baseoligonucleotidesequence.TheGCcontentshouldbebetween40%and60%;aGCcontentofapproximately45%isideal.

3.Sequencesthathaveatleast3AorTresiduesinpositions15–19ofthesensesequenceappeartohaveincreasedknockdownactiv-ity.

4.Check the 19-base oligonucleotide for secondary structure andlongbaseruns,bothofwhichcaninterferewithproperannealing.Eliminatecandidatesequencesthatdisplaythesecharacteristics.

5.Comparetheremainingcandidatesequencestoanappropriategenomedatabasetoidentifysequencesthatarespecificforthegeneofinterestandshownosignificanthomologytoothergenes.CandidatesequencesthatmeetthesecriteriaarepotentialshRNAtargetsites.

Tooptimizegenesilencing,wehighlyrecommendthatyoutestmorethanoneshRNAtargetsequenceforagene.WeprovideenoughRNAi-ReadypSIRENvectortoperform20ligations,whichallowsyoutoscreenforfunctionalshRNAsequenceswithinyourgeneofinterest.Youshouldtestatleast4shRNAspergene.ItmayhelptochooseshRNAtargetsthatarepositionedallalongthelengthofthegenesequencetoreducethechanceoftargetingaregionthatiseitherhighlystructuredorboundbyregulatoryproteins.

Note:You will need to design a gene-specific assay to test forthesuppressionofGeneX,ifyouhavenotalreadydoneso.SeeSectionIVforadditionalinformation.



B. Designing OligonucleotidesItisnecessarytosynthesizetwocomplementaryoligonucleotides(atopstrandandabottomstrand) foreachshRNAtargetsite.Figure4illustratestheoverallstructureoftheprototypicaloligonucleotidesequencesforuseinpSIREN.Thesequencesoftheoligonucleotidesshouldinclude:

• A 5'-BamH I restriction site overhang on the top strand and a5'-EcoR I restriction site overhang on the bottom strand.Theserestriction sites will enable directional cloning of the annealedoligonucleotidesintotheRNAi-ReadypSIRENvector

• Aguanine(G)residuelocatedjustdownstreamoftheBamHIsiteonthetopstrand(RNAPolIIIpreferstoinitiatetranscriptionwithaguanine)

• The19-baseoligonucleotidesensesequence(targetsensesequence)oftheshRNAtargetsite.

• A 7–9 nucleotide hairpin loop sequence (We typically use5'-TTCAAGAGA-3';seeSuiet al.,2002;Leeet al.,2002;Paddsionet al.,2002;Brummelkampet al.,2002;andPaulet al.,2002forothereffectiveloopsequences.)

• The19-baseoligonucleotideantisensesequence(targetantisensesequence)oftheshRNAtargetsite;ensureproperorientationforcorrectformationofthehairpinstructure(seeFigure2).

• ARNAPolIIIterminatorsequenceconsistingofa5–6nucleotidepoly(T)tract

• (Optional,butrecommended)Auniquerestrictionsiteimmediatelydownstream of the terminator sequence for restriction digestanalysistoconfirmthepresenceoftheclonedinsertNote:TheMlu I restrictionsitehasbeenengineered into theLuciferaseshRNAAnnealedOligonucleotideaswellastheNegativeControlshRNAAnnealedOligo-nucleotidesothatasimplediagnosticdigestcanbeusedtoverifythatthepSIRENvectorcontainstheshRNAoligo.Youcanengineerthissamerestrictionsiteintoyourgene-specificshRNAoligonucleotidesaslongasMluIdoesnotcutwithintheoligonucleotidesequences.

Atypicaloligonucleotidehas5basesfortherestrictionsiteatthe5'end,19basesofsensestrand,7–9basesofhairpinloop,19basesofan-tisensestrand,6basesofterminator,6basesofauniquerestrictionsite,and1basefortherestrictionsiteatthe3'end(whendigestedwithEcoRI),resultinginanoligonucleotideof63–65bases.SeeTableIIforexamplesofsenseandantisensesequencesdesignedforcertaingenes. Our comprehensive online designer tool at http://bioinfo2.clontech.com/rnaidesigner/candesigntherequiredoligonucleotidesforanysequenceinput.

V. shRNA Oligonucleotide Design continued

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3739-1 �0 Version No. PR63158�


V. shRNA Oligonucleotide Design continued

3'-GCNNNNNNNNNNNNNNNNNNNAAGTTCTCTNNNNNNNNNNNNNNNNNNNGAAAAAA-NNNNNN-CTTAA-5'

5'-GATCCGNNNNNNNNNNNNNNNNNNNTTCAAGAGANNNNNNNNNNNNNNNNNNNCT T T T T T-NNNNNN-G-3'BamH I

EcoR I

Hairpin loopTop strand

Bottom strand

Target sense sequence Target antisense sequenceTerminator Test RE

site

Figure 4. shRNA oligonucleotide sequence design.ArrowdenotesthepurineresiduerequiredforRNAPolIIItoinitiatetranscription.Thehairpinloopsequenceshownisoneofmanyfunc-tionalloopsequencesusedtogenerateshRNAs.Terminationissignaledusingapoly(T)tract.Includingauniquerestrictionsite(TestREsite)allowsconfirmationoftheclonedinsertaftertheligationandtransformationreactions.5'BamHIand3'EcoRIoverhangsarenecessaryfordirectionalcloningintoRNAi-ReadypSIRENvectors.SeeTableIIforexamplesoftargetsenseandantisensesequencesforcertaingenes.

TABLE II. EXAMPLES OF PuBLISHED TARGET SEQuENCESa

Gene Target sequenceb Sense sequence Antisense sequence Reference

β-actin aatgaagatcaagatcattgc tgaagatcaagatcattgc gcaatgatcttgatcttca Harborth et al., 2001Bcr-abl aagcagagttcaaagccctt gcagagttcaaagccctt aagggctttgaactctgc Scherret al., 2002hRad9 aagtctttcctgtctgtcttt gtctttcctgtctgtcttt aaagacagacaggaaagac H i ra i &Wang, 2002a Sequencesareshownfortopstrandoligodesign.Allsequencesshown5'to3'.Bottomstrandoligodesign

(notshown)isthecomplementarysequencetothetopstrand.b Identifiedfromgenecodingsequence.

SeeSectionIVforourrecommendationtousePAGE-purifiedoligo-nucleotides. It ispossibletoclonewithoutPAGEpurification,but itislikelythattheoverallligationefficiencyandthenumberofcorrectcloneswilldecreaseduetotheimpactofincompleteoligonucleotideextensions.IftheoligonucleotidesarePAGEpurified,orderatthe200nmolscale.There is no need to order phosphorylated oligonucleotides. RNAi-ReadypSIRENVectorshavenotbeendephosphorylatedafterlinear-ization;thusligationwillproceedsmoothlyusingunphosphorylatedoligonucleotides.

Protocol No. PT3739-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR63158� �1


VI. Cloning into RNAi-Ready pSIREN Vectors

A. Annealing the OligonucleotidesForconvenience,Steps3–6canbedoneinathermalcycler.

1.ResuspendeachpurifiedoligonucleotideinTEbuffertoaconcen-trationof100µM.

2.Mixtheoligosforthetopstrandandthebottomstrandata1:1ratio.Thiswillultimatelygive50µMofdsoligo(assuming100%theoreticalannealing).

3.Heatthemixtureto95°Cfor30sectoremoveallsecondarystruc-ture.Note:Heatingto95°Censuresthattheinternalhairpinofeacholigonucleotideisdisruptedandpromotesintermolecularannealing.

4.Heatat72°Cfor2min. 5.Heatat37°Cfor2min. 6.Heatat25°Cfor2min. 7.Storeonice.

TheannealedoligonucleotideisnowreadyforligationintoanRNAi-ReadypSIRENvector.Alternatively,thedsoligonucleotidecanbestoredat–20°Cuntilreadytouse.

B. Ligating ds Oligonucleotide into RNAi-Ready pSIREN 1.Dilutetheannealedoligo(fromSectionVI.A.7)withTEbufferto

obtainaconcentrationof0.5µM.Note:Toensuregoodligationefficiencyitisnecessarytodilutetheoligosothatitisonlyinmoderateexcess.Usinganexcessoftheoligowillblockligation.

2.Assemblealigationreactionforeachexperimentalannealedoligo-nucleotide.AlsosetupligationsusingtheLuciferaseshRNAandNegativeControlshRNAAnnealedOligonucleotides.

a.Foreachligation,combinethefollowingreagentsinanmicrofugetube:

2 µl LinearizedpSIRENvector(25ng/µl) 1 µl Diluted,annealedoligonucleotide(0.5µM) 1.5 µl 10XT4DNALigaseBuffer 0.5 µl BSA(10mg/ml) 9.5 µl Nuclease-freeH2O 0.5 µl T4DNAligase(400U/µl) 15 µl Totalvolume b.Setupseparateligationsusing1µloftheLuciferaseshRNAAn-

nealedOligonucleotideor1µloftheNegativeControlshRNAAnnealed Oligonucleotide. If desired, an additional controlligation(vectoronlyplusligase)canbeassembledusing1µlNuclease-freeH2Oinsteadofannealedoligonucleotide.

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3739-1 �� Version No. PR63158�


VI. Cloning into RNAi-Ready pSIREN Vectors continued

Note:The"vectoronlyplusligase"controlplateoftenyieldsnumbersofcoloniessimilartothoseobtainedfromthetestreactionsthatcombinevectorwithashRNA-annealedoligonucleotideinthepresenceofligase.DONOTBEALARMED.WebelievethattheexcessofshRNAoligopresentinthetestreactionsinhibitsreliga-tionofsinglecutvector.SincenoshRNAoligoispresentinthecontrolreactions,singlecutvectorcanreligate.Evenityouobservemanycoloniesonthe"vectoronlyplusligase"plate,proceedtoscreenthecoloniesonthetestreactionplates,sinceapproximately80%oftheseshouldcontainaninsert.

3.Incubatethereactionmixturefor3hratroomtemperature.Note: Donotlettheligationreactiongolongerthan3hr.Ifyouareunabletoim-mediatelyperformthetransformationafterthisstep,storethecompletedligationat–20°Cuntilreadytouse.

C. Transforming Fusion-Blue™ Competent Cellswith recombinant pSIRENFusion-BlueCompetentCellsareanE. coliK-12strainthatprovideshightransformationefficiency.ThestraincarriesrecAandendAmutationsthatmakeitagoodhostforobtaininghighyieldsofplasmidDNA.WeroutinelyusethisstrainforallourshRNAcloning.

1.Thawtherequirednumberoftubesofcellsonicefor10min.Tapgentlytoensurethatthecellsaresuspended.

2.Add2µloftheligationmixture(fromSectionVI.B.3)directlyto50µlofcellsuspension.MixgentlytoensureevendistributionoftheDNAsolution.

3.Incubate the transformation mixture (DNA + cells) on ice for 5min.

4.Heatthetubesforprecisely30secinawaterbathat42°Cwithoutshaking.

5.Removethetubesfromthewaterbathandplacethemdirectlyonicefor2min.

6.Add250µlroom-temperatureSOCmediumtoeachtube.Incubateat37°Cfor60minwhileshakingat250rpm.

7.Plate30µl(1/10ofthetransformationvolume)fromeachtransforma-tiononselectivemediumcontainingtheappropriateconcentrationofantibiotic.Incubateat37°C.Notes

• Bothcellcompetencyandligationefficiencyaffecttheoutcomeofthetrans-formation.Wesuggestplatingdifferentamountsonseparateplatestoidentifythe optimal volume for determining transformation efficiency and isolatingcolonies.

• Platingisaccomplishedbyspreadingcellsonselectivemedium[e.g.,LBagar+Ampicillin(50–100µg/ml)].SeetheProductAnalysisCertificatethataccompaniestheRNAi-ReadypSIRENvector.

• WehaveobservedthatrecombinantpSIRENDNAusuallygeneratessmallerandslower-growingcolonies.



VI. Cloning into RNAi-Ready pSIREN Vectors continued

• Iftheplatingdensityistoohigh,dilutethetransformationmixture1:10withroom-temperatureSOCandspreadplateswith30µlofthe1:10dilution(1/100oftheoriginaltransformationvolume).

8.Inoculateasmall-scaleliquidculturewithasingle,well-isolatedcolony.Werecommendyousetup4–8suchculturestoensureyouobtainatleastonepositiveclone.Afterovernightincubation,isolateplasmidDNAusinganystandardmethod.Forsmall-scalepurification(20µgplasmidDNA),werecommendourNucleoSpinPlasmidKit(Cat.No.635988).

9.IdentifythedesiredrecombinantplasmidbyrestrictionanalysisusingtheuniquerestrictionsitewithintheshRNAoligonucleotidesequence.Ifdesired,verifyyourinsertbysequencing.Notes:TheMluIrestrictionsitehasbeenengineeredintotheLuciferaseshRNAAnnealedOligonucleotideaswellastheNegativeControlshRNAAnnealedOligo-nucleotidesothatasimplediagnosticdigestcanbeusedtoverifythatthepSIRENvectorcontainstheshRNAoligo.

Sincethereisalwaysachanceformutationsintheoligoduetosynthesiserrors,westronglyrecommendthatyousequenceatleasttwoclonestoverifythecorrectoligosequence.Becausehairpinsequencesaredifficulttosequence,informyoursequencingfacilitysothatsequencingconditionscanbeadjustedaccordingly.

10.Onceapositiveclonehasbeenidentified,inoculatealarge-scaleliquidculturetopreparegreaterquantitiesofyourrecombinantpSIRENvector.Toensureoptimalpurityof theDNA, isolateallplasmidsfortransfectionusingaNucleoBondPlasmidMaxiEFKit(Cat.No.635953)orbyCsCldensitygradientpurification(Sambrook&Russell,2001).

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3739-1 �� Version No. PR63158�


VII. Transfection of Recombinant pSIREN Vectors

ThetransfectionprotocolincludedinthisUserManualisintendedforboththescreeningforfunctionalshRNAconstructsandgenesilencingexperi-mentsusingtransfection.Ifyouwillperformexperimentsusingviraldelivery,firstusethistransfectionproceduretoscreenconstructs,thenproceedtothesecondaryUserManualsincludedwithyourKnockoutRNAiSystem.ThesecondaryUserManualsdescribetheprotocolsforviralpackagingandinfectionusingpSIRENconstructs.Ifyourtargetcellscannotbetransfected,thenviraldeliveryshouldbetriedforbothfunctionalshRNAscreeningandgenesilencingexperiments.

Forfurtherinformationoncellculturetechniques,seeFreshney(2000).

Theefficiencyofamammaliantransfectionprocedureisprimarilydependentonthehostcellline.Therefore,whenworkingwithacelllineforthefirsttime,werecommendyoucomparetheefficienciesofseveraltransfectionprotocols.Afterchoosingamethodoftransfection,optimizecelldensity(usually60–80%confluency), theamountandpurityof theDNA,mediaconditions,andtransfectiontime.

If a transfectionmethod is alreadyestablished for your cell linemodel,proceedwiththoseconditions.Itisimportanttokeepoptimizedparametersconstanttoobtainreproducibleresults.

Forourtransfections,wehavebeensuccessfulusingtheCalPhos™Mam-malianTransfectionKit(Cat.No.631312)andtheCLONfectin™TransfectionReagent(Cat.No.631301).

Thefollowingprotocol isdesignedforusewithCLONfectinTransfectionReagentusingadherentculturesin6-welltissue-cultureplates.Ifyouareusingothervessels,adjust thecomponents inproportionto thesurfaceareaofyourplate/flask.SeetheCLONfectinUserManual(PT3005-1),Ap-pendixBforcultureplateconversions.Perform all steps in a sterile tissue culture hood.

1.Platecellstwodaysbeforethetransfectionexperiment;theyshouldbe60–70%confluentonthedayoftransfection.Wegenerallyplate1–4x105cells/well.

2.Incubateplatesat37°CinaCO2incubatorfor24–48hr. 3.Onthedayofthetransfection,preparefreshliposomesolution: a. WarmHEPES-BufferedSaline(HBS)to45–55°C. b. Add 90 µl of HBS dropwise to a 100-µg aliquot of

CLONfectin Reagent stock to make a final concentration of1µg/µl.

c. Immediatelyvortexgently. d. Placeonice.



4.Foreachtransfection,preparesolutionAandsolutionBinseparate,sterilepolystyrenetubes.

Solution A: 2–4µg plasmidDNA 100µl Serum-freemedium

Solution B: 2–8µg CLONfectinReagent(1µg/µlinHBS) 100µl Serum-freemedium

Notes: • A1:1(w/w)ratioofDNA:CLONfectinReagentworksbestformanycelltypes. • Toreducevariabilitywhentransfectingmultiplewellswiththesameplasmid

DNA,prepareadequatevolumesofsolutionsAandBforallwells. • YoumaystoreunusedCLONfectinReagentat4°Cuptooneweek.

5.CombinesolutionsAandBintoonetubeandmixgently. 6.Incubatethetransfectionsolutionatroomtemperaturefor10–30

min. 7.Add 1.8 ml of serum-free medium to each tube containing the

CLONfectinReagent/DNAsolutionandmixgently. 8.Remove themediumfromthecultures tobe transfected,wash

withPBS,andgentlyapplytheCLONfectinReagent/DNA/mediasolution.

Notes: •Washingthecellswithserum-freemediumbeforeapplyingtheCLONfectin/DNA

solutionisnotnecessary.Anyserumpresentwillbeverydilute(<1%)andwillnotaffecttransfectionsusingCLONfectinReagent.Youmayaddserumtoafinalconcentrationof2.5%totheCLONfectinReagent/DNA-containingmediumatthispointtoimprovecellviability.

• Donotaddantibacterialorantifungalagents to themediumduring transfec-tion.

9.Gentlyrockplatesbackandforthtodistributetransfectionsolutionevenly.Donotrotateplatesasdoingsowillconcentratetransfec-tionprecipitateinthecenterofthewell.

10.Incubateplatesat37°Cfor4–6hrinaCO2incubator. 11.RemoveCLONfectinReagent/DNA-containingmediumandwash

cellswithmediumor1XPBS.PrewarmmediaorPBSto37°C. 12.Apply2mloffreshcompletegrowthmediumandincubateat37°C

untilcellsareneededforassay. 13.Assay for transient gene suppression 48 hr post-transfec-

tion.Note:IfyouhavetransfectedapSIREN-RetroQconstruct,youcanstartselectionforstabletransformants48–72hrpost-transfection.

VII. Transfection of Recombinant pSIREN Vectors cont.



Figure 5. Procedures provided for viral delivery of recombinant pSIREN.Aftergeneratingfunc-tionalpSIRENconstructsusingtheproceduredetailedinthisUserManual,proceedwiththeprotocolsdescribedintheprovidedviralexpressionUserManualstoproducerecombinantvirusforefficientdeliveryofrecombinantpSIRENtomanycelltypes.

VIII. Viral Delivery of pSIREN Constructs

Knockout™RNAi SystemsUser Manual

(PT3739-1)

RNAi-Ready pSIREN-RetroQ Vector

(Cat. No. 631526)*

Knockout™ Adenoviral RNAi System 1

(Cat. No. 631528)

Knockout™ Adenoviral RNAi System 2

(Cat. No. 631529)

Retroviral Gene Transfer and Expression User Manual

(PT3132-1)Start with "Virus Production"

* The RNAi-Ready pSIREN-RetroQ-ZsGreen Vector (Cat. No. 632455) or RNAi-Ready pSIREN-RetroQ-DsRed-Express Vector (Cat. No. 632487) may be used instead of the pSIREN-RetroQ Vector, for the production of recombinant pSIREN retroviral particles expressing a fluorescent marker.

Adeno-X™ Expression System 1 User Manual

(PT3414-1)Start with "PI-Sce I / I-Ceu I

Digestion ..."

Adeno-X™ Expression System 1 User Manual

(PT3414-1)

Adeno-X™ Expression Systems 2 User Manual

(PT3674-1)Start with Creator™

Adenoviral DNA Cloning Protocol"

Knockout™ RNAi SystemsUser Manual

(PT3739-1)

Knockout ™RNAi SystemsUser Manual

(PT3739-1)

Retroviral Adenoviral

OnceyouhaveidentifiedfunctionalpSIRENconstructsforspecificgenesilencing,youcanproceedwithviralpackagingoftheconstructs.Forthispurpose,secondaryUserManualsaresuppliedwithyourRNAiSystem.Figure5illustratestheproceduralstepsforretroviralandadenoviraldelivery.



IX. Analysis of Results and Troubleshooting Guide

A. Poor transformation efficiencyLowtransformationefficiencycanbetheresultofaproblemwiththeoligonucleotides,ligation,and/ortransformation.Incompatibleends Confirm that the ends of the annealed oligo-ontheinsert nucleotidecontain5'BamHIand3'EcoRIover-

hangsforproperligationintopSIREN.

Ineffectiveoligo Verifythatthetopandbottomstrandsequencesannealing arecorrect.ToensureahighamountofdsDNAin

theannealingreaction,mixanequalratiooftopandbottomstrands.Itmaybenecessarytoincreasethedenaturationtemperature(SectionVI.A.3.)toincreasetheyieldofannealedoligonucleotide.

Oligosarenot Verifyoligonucleotidesizeusinga12%nativefull-length polyacrylamide gel. Order PAGE-purified oligo-

nucleotidestoensureahigherpercentageoffull-lengtholigonucleotidesandincreasethechanceofcloningacompleteandfunctionalinsert.

Suboptimaloligo Verifytheconcentrationoftheannealedoligoconcentrationin nucleotideusedforligation.Toolittleortooligation mucholigonucleotidecanaffectligation.Toim-

proveligationefficiency,performarangeof5-or10-folddilutionsoftheannealedoligonucleotideforuseinligation.

Inactiveligase Checkyourligaseandligasebufferforactivityand/orligase usingadifferentvectorandinsert.Replacebuffer theligationreagentsiftheyproveinactive.

Suboptimal TransformFusion-BlueCompetentCellsusingcompetentcells theprovidedTestPlasmid.Calculatethenumberof

cfu/µgtodeterminethecells'competency.Handlecompetentcellsgentlyduringtransformationandplating.

Performtheheatshockstep(StepVI.C.4.)forpre-cisely30sec.Extendingthistimewilldrasticallyreducecellviability.

Wrongantibiotic Verifythecorrectantibioticanditsorsuboptimalanti- concentrationbycheckingtheProductAnalysisbioticconcentration Certificate that accompanies the RNAi-Ready

pSIRENvector.



B. Poor transfection efficiencyTransfectionefficiencycanbeaffectedbyplasmidpurityortransfec-tionconditions.Alternatively,anineffectivepSIRENconstructcanbemisinterpretedaslowtransfectionefficiency.

Poorpurity EnsurethepurityofrecombinantpSIRENDNAbyofthepSIREN isolatingallplasmidsfortransfectionusingaconstruct NucleoBondPlasmidMidiKit (Cat.No.635931)

orbyCsClgradient.

Ineffective Theefficiencyofamammaliancelltransfectiontransfection dependsprimarilyonthehostcellline.Optimiz-

ingthetransfectionparametersforeachcelltypeis crucial to obtaining consistently successfultransfections.Therefore, for each cell type youplantouse,performpreliminaryexperimentstodeterminetheoptimal:1)amountoftransfectionreagent;2)amountandpurityofDNA;3)ratiooftransfection reagent to DNA; 4) cell density; 5)transfectionincubationtime;and6)mediacon-ditions.IfyouareusingCLONfectinTransfectionReagent,seetheUserManualformoreinforma-tion.

Nodetectable Youshouldtestatleast3-4pSIRENconstructsgenesilencing pergenetooptimizegenesilencing.Weprovide

enoughRNAi-ReadypSIRENvectorineachkittoperform20ligations,whichallowsyoutoscreenforfunctionalshRNAsequenceswithinyourgeneofinterest.

We have not observed loss or mutation of theannealedoligonucleotideswhenclonedintoRNAi-ReadypSIRENvectorsandpropagatedusingtherecommendedconditions.Toensureintegritydonotovergrowtransformedcultures.Ifplanninganovernightculture,inoculateaslateaspossibleinthedayusinga1:1000dilutionoffreshlygrownstock.Incubatewithsufficientshakingtoensuregoodaeration(250rpm)andharvestthecultureasearlyaspossiblethenextdaytopreventcul-ture overgrowth. Do not serially passage yourcultures.Inadditiontokeepingglycerolstocksoftransformedcells,wehighlyrecommendkeepingDNAstocksofyourpSIRENconstructs.

IX. Analysis of Results and Troubleshooting... cont.



X. References

Aaronson,J.S.,Eckman,B.,Blevins,R.A.,Borkowski,J.A.,Myerson,J.,Imran,S.&Elliston,K.O.(1996)Towardthedevelopmentofageneindextothehumangenome:Anassessmentofthenatureofhigh-throughputESTsequencedata.Genome Res.6:829–845.

Abremski,K.&Hoess,R.(1984)BacteriophageP1site-specificrecombination.PurificationandpropertiesoftheCrerecombinaseprotein.J. Biol. Chem. 259:1509–1514.

Adeno-XExpressionSystem(January2000)Clontechniques XV (1):8–10.

BDRetro-XªQVectors.(July2002)Clontechniques XVII(3):13–14.

Bernstein,E.,Caudy,A.A.,Hammond,S.A.,&Hannon,G.J.(2001)Roleforabidentateribo-nucleaseintheinitiationstepofRNAinterference.Nature 409:363–366.

Brummelkamp,T.R.,Bernards,R.&Agami,R.(2002).AsystemforstableexpressionofshortinterferingRNAsinmammaliancells.Science 296:550–553.

Chase,D.,Serafinas,C.,Ashcroft,N.,Kosinski,M.,Longo,D.,Ferris,D.K.&Golden,A.(2000)Thepolo-likekinasePLK-1isrequiredfornuclearenvelopebreakdownandthecompletionofmeiosisinCaenorhabditis elegans.Genesis 26:26–41.

Elbashir,S.M.,Lendeckel,W.&Tuschl,T.(2001)RNAinterferenceismediatedby21-and22-nucleotideRNAs.Genes Dev.15(2):188–200.

Emerman,M.&Temin,H.M.(1984)Geneswithpromotersinretrovirusvectorscanbeinde-pendentlysuppressedbyanepigeneticmechanism.Cell 39:449–467.

Fire,A.,Xu,S.,Montgomery,M.K.,Kostas,S.A.,Driver,S.E.&MelloC.C.(1998)Potentandspecificgeneticinterferencebydouble-strandedRNAinCaenorhabditis elegans.Nature 391:806–811.

Freshney,R.I.(2000)CultureofAnimalCells,FourthEdition(Wiley-Liss,NY).

Gonczy,P.,Pichler,S.,Kirkham,M.&Hyman,A.A.(1999)CytoplasmicdyneinisrequiredfordistinctaspectsofMTOCpositioning,includingcentrosomeseparation,intheonecellstageCaenorhabditiselegansembryo.J. Cell Biol. 147:135–150.

Guo,S.&Kempheus,K.J.(1995)Par-1,agenerequiredforestablishingpolarityinC. elegansem-bryos,encodesaputativeSer/Thrkinasethatisasymmetricallydistributed.Cell 81:611–620.

Hammond,S.M.,Caudy,A.A.&Hannon,G.J.(2001)Post-transcriptionalGeneSilencingbyDouble-strandedRNA. Nature Rev. Gen.2:110–119.

Harborth, J., Elbashir, S. M., Bechert, K.,Tuschl,T. &Weber, K. (2001) Identification of es-sential genes in cultured mammalian cells using small interfering RNAs. J. Cell Science 114:4557–4565.

Hillier,L.,et al.(1996)Generationandanalysisof280,000humanexpressedsequencetags.Genome Res.6:807–828.

Hirai,H.&WangH-G.(2002)AroleoftheC-terminalregionofhumanRad9(hRad9)innucleartransportofthehRad9checkpointcomplex.J. Biol. Chem.277(28):25722–25727.

Hutvagner,G.&Zamore,P.D.(2002)RNAi:natureabhorsadouble-strand.Curr. Opin. Genet-ics & Development.12:225–232.

Jantsch-Plunger,V.&Glotzer,M.(1999)DepletionofsyntaxinsintheearlyCaenorhabditis elegansembryorevealsaroleformembranefusioneventsincytokinesis.Curr. Biol.9:738–745.

Julius,M.A.,Yan,Q.,Zheng,Z.&Kitajewski,J.(2000)QVectors,Bicistronicretroviralvectorsforgenetransfer.BioTechniques28(4):702–707.



Kennerdell,J.R.&Carthew,R.W.(1998)UseofdsRNA-mediatedgeneticinterferencetodem-onstratethatfrizzledandfrizzled2actinthewinglesspathway.Cell 95:1017–1026.

Kinsella,T.M.&NolanG.P. (1996)Episomalvectors rapidlyandstablyproducehigh-titerrecombinantretrovirus.Hum. Gene Ther. 7:1405–1413.

Kunkel,G.R.&Pederson,T.(1989)TranscriptionofahumanU6smallnuclearRNAgenein vivowithstandsdeletionofintragenicsequencesbutnotofanupstreamTATATAbox.Nucl. Acids Res.17:7371–7379.

Lee,N.S.,Dohjima,T.,Bauer,G.,Li,H.,Li,M-J.,Ehsani,A.,Salvaterra,P.&Rossi,J.(2002)ExpressionofsmallinterferingRNAstargetedagainstHIV-1revtranscriptsinhumancells.Nature Biotechnol.20:500–505.

Matz,M.V.,Fradkov,A.F.,Labas,Y.A.,Savitsky,A.P.,Zaraisky,A.G.,Markelov,M.L.&Lukya-nov, S.A. (1999) Fluorescent proteins from nonbioluminescentAnthozoa species. Nature Biotech.17:969–973.

Manche,L.,Green,S.R.,Schmedt,C.&Mathews,M.B.(1992)Interactionsbetweendouble-strandedRNAregulatorsandtheproteinkinaseDAI.Mol. Cell. Biol.12:5238–5248.

Minks,M.A.,West,D.K.,Benvin,S.&Baglioni,C.(1979)Structuralrequirementsofdouble-strandedRNAfortheactivationof2'-5'-oligo(A)polymeraseandproteinkinaseofinterferon-treatedHeLacells.J. Biol. Chem.254:10180–10183.

Mizuguchi,H.&Kay,M.A.(1999)AsimplemethodforconstructingE1-andE1/E4-deletedrecombinantadenoviralvectors.Hum. Gene Ther. 10:2013–2017.

Moore,L.L.,Morrison,M.&Roth,M.B.(1999)HCP-1,aproteininvolvedinchromosomeseg-regation,islocalizedtothecentromereofmitoticchromosomesinCaenorhabditis elegans. J. Cell Biol.147:471–480.

Napoli,C.,Lemieux,C.&Jorgensen,R.(1990)IntroductionofaChimericChalconeSynthaseGeneintoPetuniaResultsinReversibleCo-SuppressionofHomologousGenesintrans. Plant Cell2:279–289.

Nykanen,A.,Haley,B.&Zamore,P.D.(2001)ATPrequirementsandsmallinterferingRNAstructureintheRNAinterferencepathway.Cell 107:309–321.

Ory,D.S.,Neugeboren,B.A.&Mulligan,R.C.(1996)Astablehuman-derivedpackagingcelllineforproductionofhightiterretrovirus/vesicularstomatitisvirusGpseudotypes.Proc. Natl. Acad. Sci. USA93:11400–11406.

Paddison,P.J.,Caudy,A.A.,Bernstein,E.,Hannon,G.J.&Conklin,D.S.(2002).Shorthair-pinRNAs (shRNAs) inducesequence-specificsilencing inmammaliancells.Genes & Dev.16:948–958.

Paul,C.P.,Good,P.D.,Winer,I.&Engelke,D.R.(2002)EffectiveexpressionofsmallinterferingRNAinhumancells.Nature Biotechnol.20:505–508.

Pear,W.S.,Nolan,G.P.,Scott,M.L.&Baltimore,D.(1993)Productionofhigh-titerhelper-freeretrovirusesbytransienttransfection.Proc. Natl. Acad. Sci. USA90(18):8392–8396.

Sambrook,J.&Russell,D.(2001)MolecularCloning:ALaboratoryManual,3rdEdition,(ColdSpringHarborLaboratoryPress,ColdSpringHarbor,NY).

Sauer, B. (1994) Site-specific recombination: developments and applications. Curr. Opin. Biotechnol. 5:521–527.

Scherr,M.,Battmer,K.,Winkler,T.,Heidenreich,O.,Ganser,A.&Eder,M.(2002)Specificinhibi-tionofbcr-ablgeneexpressionbysmallinterferingRNA.Blood 101(4):1566–1569.

X. References continued



Schumacher,J.M.,Golden,A.&Donovan,P.J.(1998)AIR-2:AnAurora/Ipl1-relatedproteinkinaseassociatedwithchromosomesandmidbodymicrotubulesisrequiredforpolarbodyextrusionandcytokinesisinCaenorhabditis elegansembryos.J. Cell Biol.143:1635–1646.

Sedivy,J.M.&Dutriaux,A.(1999)GenetargetingandsomaticcellgeneticsÑarebirthoracomingofage?Trends Genet.15(3):88–90.

Sharp,P.A.(2001)RNAInterferenceÑ2001.Genes Dev.15:485–490.

Sui,G.,Soohoo,C.,Affar,E.B.,Gay,F.,Shi,Y.,Forrester,W.C.&Shi,Y.(2002)ADNAvector-basedRNAitechnologytosuppressgeneexpressioninmammaliancells.Proc. Natl. Acad. Sci. USA99(8):5515–5520.

Yang,S.,Delgado,R.,King,S.R.,Woffendin,C.,Barker,C.S.,Yang,Z.Y.,Xu,L.,Nolan,G.P.&Nabel,G.J.(1999)Generationofretroviralvectorforclinicalstudiesusingtransienttransfec-tion.Hum. Gene Ther. 10:123–132.

Yu,J-Y.,DeRuiter,S.L.&Turner,D.L.(2002)RNAinterferencebyexpressionofshort-interferingRNAsandhairpinRNAsinmammaliancells.Proc. Natl. Acad. Sci. USA99(9):6047–6052.

X. References continued



XI. Related Products

ForacompletelistingofallClontechproducts,pleasevisitwww.clontech.com

Cat. No. • Fusion-Blue™CompetentCells 636700

• CLONfectin™TransfectionReagent 631301 631302

• CalPhos™MammalianTransfectionKit 631312

• Adeno-X™ExpressionSystem1 631513

• Adeno-X™System1ViralDNA 631026 (linear)

• Adeno-X™AccessoryKit 631027

• Adeno-X™PCRPrimerScreeningSet 631030

• Adeno-X™ExpressionSystem2 631524

• Adeno-X™PromoterlessExpression 631525 System2

• Adeno-X™VirusPurificationKit 631532

• Adeno-X™VirusPurificationKit 631533

• Adeno-X™VirusPurificationMegaKit 631534

• Adeno-X™RapidTiterKit 631028

• Adeno-X-DsRed2Adenovirus 632417

• Adeno-X-LacZAdenovirus 631029

• Adeno-X-NullAdenovirus 631517

• Retro-X™System 631508

• Retro-X™UniversalPackagingSystem 631530

• PantropicRetroviralExpressionSystem 631512

• RetroPack™PT67CellLine 631510

• AmphoPack™-293CellLine 631505

• EcoPack™2CellLine 631507



XI. Related Products continued

Cat. No. • NucleoSpin®Multi-8PlusPlasmidKit 635976

• NucleoBond®PlasmidMaxiEFKit 635953

• NucleoBond®PlasmidMidiKit 635931

• PowerScript™ReverseTranscriptase 639500 639501

• Advantage®RT-for-PCRKit 639505 639506

• TITANIUM™TaqDNAPolymerase 639208 639209



Appendix: Vector Information

5'-TGTGGAAAGGACGAGGATCC[...shRNA oligo cloning site...]GAATTCTACCGGGTAGGGGAGGCGCT T TTCCCAAGGCAGT-3'

3'-ACACCTTTCCTGCTCCTAGG[...shRNA oligo cloning site...]CTTAAGATGGCCCATCCCCTCCGCGAAAAGGGTTCCGTCA-5'

6440•

6430•

10•

BamH I EcoR I

20•

30•

40•

Figure 6. Restriction Map and Cloning Site of the RNAi-Ready pSIREN-RetroQ Retroviral Vector. Uniquerestrictionsitesareinbold.RNAi-ReadypSIREN-RetroQisprovidedasalinearizedvectordigestedwithBamHIandEcoRI.Nucleotidesingraywereremovedduringlineariza-tion.ThislinearizedvectorisreadyforligationofadsDNAoligonucleotidecontainingBamHIandEcoRIoverhangs.RNAi-ReadypSIREN-RetroQisaself-inactivatingretroviralexpres-sionvectordesignedtoexpressasmallhairpinRNA(shRNA)usingthehumanU6promoter(PU6;RNAPolIII-dependent).ThisvectorisusedfortargetedgenesilencingwhenadsDNAoligonucleotideencodinganappropriateshRNAisligatedintothevector.YoucantransfectyourpSIREN-RetroQconstructasaplasmidexpressionvector,orupontransfectionintoapackagingcellline,thisvectorcantransientlyexpress,orintegrateandstablyexpressaviralgenomictranscriptcontainingthehumanU6promoterandtheshRNA.Thevectorcontainsapuromycinresistancegenefortheselectionofstabletransfectants.Thisretroviralvectorisoptimizedtoeliminatepromoterinterferencethroughself-inactivation.Thehybrid5'LTRconsistsofthecytomegalovirus(CMV)typeIenhancerandthemousesarcomavirus(MSV)promoter.ThisconstructdriveshighlevelsoftranscriptioninHEK293-basedpackagingcelllinesdue,inpart,tothepresenceofadenoviralE1A(Kinsella&Nolan,1996;Oryet al.,1996;Pearet al.,1993;andYanget al.,1999)inthesecells.Theself-inactivatingfeatureofthevectorisprovidedbyadeletioninthe3'LTRenhancerregion(U3).DuringreversetranscriptionoftheretroviralRNA,theinactivated3'LTRiscopiedandreplacesthe5'LTR,resultingininactivationofthe5'LTRCMVenhancersequences.Thismayreducethephenomenonknownaspromoterinterference(Emerman&Temin,1984)andallowmoreefficientexpression.AlsoincludedintheviralgenomictranscriptarethenecessaryviralRNAprocessingelementsincludingtheLTRs,packagingsignal(Psi+),andtRNAprimerbindingsite.pSIREN-RetroQalsocontainsabacterialoriginofreplicationandE. coliAmprgeneforpropagationandselectioninbacteria.

RNAi-Ready pSIREN-RetroQ-ZsGreenandRNAi-Ready pSIREN-RetroQ-DsRed-Express consti-tutivelyexpressaZoanthus sp.greenfluorescentprotein(ZsGreen)andavariantofDiscosoma sp.redfluorescentprotein(DsRed-Express),respectively(Matzet al.,1999).Thefluorescentmarkerallowsyoutodirectlymonitorthedeliveryefficiencyofyourgenesilencingconstructusingeitherfluorescencemicroscopyorflowcytometry.CompletesequenceinformationisprovidedintheRNAi-ReadypSIREN-RetroQ-ZsGreenVectorInformationPacket(PT3777-5)andtheRNAi-ReadypSIREN-RetroQ-DsRed-ExpressVectorInformationPacket(PT3830-5).

RNAi-ReadypSIREN-RetroQ

6.4 kb

3'LTR

CMV/MSV5' LTR

Ψ+

Ampr

Puror

ColE1 oriSV40 ori

PSV40

PU6

PPGKBamH I (6441)

EcoR I (2)



Appendix: Vector Information continued

5'-TGGAAAGGACGAGGATCC[...shRNA oligo cloning site...]GAATTCATCTATGTCGGGTGCGGAGAAAGAGGTAATGAAAT-3'

3'-ACCT T TCCTGCTCCTAGG[...shRNA oligo cloning site...]CTTAAGTAGATACAGCCCACGCCTCT T TCTCCATTACTTTA-5'

3140•

3130•

BamH I EcoR I

10•

20•

30•

40•

Figure 7. Restriction Map and Cloning Site of the RNAi-Ready pSIREN-Shuttle Vector. Uniquerestrictionsitesareinbold.RNAi-ReadypSIREN-ShuttleisprovidedasalinearizedvectordigestedwithBamHIandEcoRI.Nucleotidesingraywereremovedduringlinearization.ThislinearizedvectorisreadyforligationofadsDNAoligonucleotidecontainingBamHIandEcoRIoverhangs.RNAi-ReadypSIREN-Shuttleisavailableseparately,andasacomponentoftheKnockoutAd-enoviralRNAiSystem1(Cat.No.631528).RNAi-ReadypSIREN-ShuttleisanexpressionvectordesignedtoexpressansmallhairpinRNA(shRNA)usingthehumanU6promoter(PU6;RNAPolIII-dependent).ThisvectorisusedfortargetedgenesilencingwhenadsDNAoligonucleotideencodinganappropriateshRNAisligatedintothevector.ThepSIREN-Shuttleexpressioncas-setteconsistsofthehumanU6promoter(PU6)andacloningsite.TheentirecassetteisflankedbyuniqueI-CeuIandPI-Sce IrestrictionsitessothatitcanbeexcisedanddirectlyligatedtoAdeno-XViralDNA,theadenoviralgenome.ThevectorbackbonealsocontainsthepUCorigin(pUCori)andakanamycinresistancegene(Kanr)forpropagationandselectioninE. coli.YoucantransfectyourpSIREN-Shuttleconstructasaplasmidexpressionvector,orinconjunctionwiththeAdeno-X™ExpressionSystem1(Cat.No.631513;January2000Clontechniques)oritsAccessoryKits(Cat.Nos.631026&631027);thisvectorcantransientlyexpressaviralgenomictranscriptcontainingthehumanU6promoterandtheshRNA.IntheAdeno-XSystem,standardligationtechniquesareusedtotransferanexpressioncassettefrompSIREN-Shuttletoareplication-deficient,Ad5genome(Mizuguchi&Kay,1999).

RNAi-ReadypSIREN-Shuttle

3.1 kb

PU6

Kanr

pUC ori

PI-Sce I (22)

BamH I (3142)

EcoR I (2)

I-Ceu I (2869)



Appendix: Vector Information continued

RNAi-ReadypSIREN-DNR

5.1 kb

pUC ori

Ampr

SacB

loxP

Cmr

(ORF)

loxP

Not I (4828)

Not I (938)

EcoR I (2)

BamH I (5129)

PU6

5'-ATATATCTTGTGGAAAGGACGAGGATCC[...shRNA oligo cloning site...]GAATTCTCTAGACCATTCGTT TGGCGCGCGG-3'

3'-TATATAGAACACCT T TCCTGCTCCTAGG[...shRNA oligo cloning site...]CT TAAGAGATCTGGTAAGCAAACCGCGCGCC-5'EcoR IBamH I

5110•

5120•

5130•

10•

20•

30•

Figure 8. Restriction Map and Cloning Site of the RNAi-Ready pSIREN-DNR Vector. Uniquerestrictionsitesareinbold.RNAi-ReadypSIREN-DNRisprovidedasalinearizedvectordi-gestedwithBamHIandEcoRI.Nucleotidesingraywereremovedduringlinearization.Thislinearizedvector is ready for ligationofadsDNAoligonucleotidecontainingBamH Iand EcoRIoverhangs.TheRNAi-ReadypSIREN-DNRisprovidedasacomponentoftheKnockoutAdenoviralSystem2(Cat.No.631529).RNAi-ReadypSIREN-DNRisdesignedtoexpressasmallhairpinRNA(shRNA)drivenbythehumanU6promoter(PU6;RNAPolIII-dependent).ThisvectorisusedfortargetedgenesilencingwhenadsDNAoligonucleotideencodinganappropriateshRNA is ligated into thevector.pSIREN-DNRhasbeendesignedasaDonorVector to transfer the shRNA expression cassette (SEC) to the adenoviralAcceptorVectorpLP-Adeno-X-PRLSViralDNAbyCre-loxPmediatedrecombination.Inaddition,pSIREN-DNRcanbeuseddirectly in transient transfectionexperiments toscreenshRNAconstructs forfunctionality.Cre,a38-kDarecombinaseproteinfrombacteriophageP1,mediatesrecombi-nationbetweenDNAsequencesatspecificlocationscalledloxPsites(Sauer,1994;Abremski&Hoess,1984).ThepSIREN-DNRVectorcontains two loxPsites,whichflankthe5'endoftheSECandthe5'endoftheopenreadingframeencodingthechloramphenicolresistancegene(Cmr).Onceyouroligonucleotideisinserted,theresultingDonorVectorconstructcanbecombinedwithanypromoterlessAcceptorVectorandCrerecombinase;CremediatesthetransferoftheSECtotheAcceptorVector.Asaresult,arecombinantplasmidiscreatedthatexpressesyourshRNAinthedesiredhostsystem.TheSEC,oncetransferred,willbecomelinkedtothespecificexpressionelementsforwhichtheAcceptorVectorwasdesigned.ForacompletelistofAcceptorVectors,visitourwebsiteatwww.clontech.com.Thisvectoralsocontainsanampicillinresistancegene,whichisthemarkerforpropagationandselectionoftheDonorVectorinE. coli.Inaddition,pSIREN-DNRcontainsthesucrasegenefromB. subtil-lis(SacB),whichprovidesnegativeselectionagainstincorrectrecombinantsandtheparentalDonorVectorfollowingrecombination.

RNAi-Ready pSIREN-DNR-DsRed-ExpressconstitutivelyexpressesavariantofDiscosoma sp.redfluorescentprotein(DsRed-Express;Matzet al.,1999).TheDsRed-Expressfluorescentmarkerallowsyoutodirectlymonitorthedeliveryefficiencyofyourgenesilencingconstructusingeitherfluorescencemicroscopyorflowcytometry.CompletesequenceinformationisprovidedintheRNAi-ReadypSIREN-DNR-DsRed-ExpressVectorInformationPacket(PT3778-5).



Notes



Notes

Notice to PurchaserThisproductisintendedtobeusedforresearchpurposesonly.Itisnottobeusedfordrugordiagnosticpurposesnorisitintendedforhumanuse.Clontechproductsmaynotberesold,modifiedforresale,orusedtomanufacturecommercialproductswithoutwrittenapprovalofClontechLaboratories,Inc.

TheCreator™TechnologyisbasedontheprocessofinvitroCre-LoxPrecombination.Clon-techistheassigneeofU.S.PatentNos.6,410,317;6,977,165;6,838,285andotherpatentspendingcoveringCreatorvectorsandtheselectionprocessasitrelatestotheproductionofrecombinantclones.Clontechhaschosennottoexerciseitsrighttoimposelicensefeesonthein-houseuseoftheCreatorTechnology.Howeveraroyalty-bearinglicenseisrequiredoncontractservicesandsaleordistributionofclonesmadeintheCreatorSystemformat.ForinformationonusingCreatorTechnologyforcommercialpurposes,pleasecontactalicensingrepresentativebyphoneat650.919.7320orbye-mailatlicensing@clontech.com.

ForLiving Colors®Products

AcGFP1,DsRed,HcRed,AsRed,AmCyan,ZsGreen,ZsYellowandtheirvariants:

TheseproductsarethesubjectsofpendingU.S.andforeignpatents.

Not-For-ProfitEntities:OrdersmaybeplacedinthenormalmannerbycontactingyourlocalrepresentativeorClontechCustomerServiceat650.919.7300.Atitsdiscretion,ClontechgrantsNot-For-ProfitEntitiesanon-exclusive,royalty-free,personal,limitedlicensetousethisproductfornon-commerciallifescienceresearchuseonly.Suchlicensespecificallyexcludestherighttosellorotherwisetransferthisproduct,itscomponentsorderivativesthereoftothirdpar-ties.NomodificationstotheproteincodingsequencemaybemadewithoutexpresswrittenpermissionfromClontech.AnyotheruseofthisproductrequiresalicensefromClontech.Forlicenseinformation,pleasecontactalicensingrepresentativebyphoneat650.919.7320orbye-mailatlicensing@clontech.com.

For-ProfitEntitieswishingtousethisproductarerequiredtoobtainalicensefromClontech.Forlicenseinformation,pleasecontactalicensingrepresentativebyphoneat650.919.7320orbye-mailatlicensing@clontech.com.

TheCMVpromoteriscoveredunderU.S.PatentNos.5,168,062,and5,385,839assignedtotheUniversityofIowaResearchFoundation.

NucleoBond®andNucleoSpin®areregisteredtrademarksofMACHEREY-NAGELGmbHandCo.,KG.Clontech,ClontechLogoandallothertrademarksarepropertyofClontechLaboratories,Inc.ClontechisaTakaraBioCompany.©2006

knockout rnai systems user manual - dartmouth collegetonyz/retroq.pdf · vii. transfection of ......

Documents