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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
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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�
Knockout™ RNAi Systems User Manual
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
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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
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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
I. Introduction continued
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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
I. Introduction continued
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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.
I. Introduction continued
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)
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I. Introduction continued
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
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I. Introduction continued
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.
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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)
• 20 µl LuciferaseshRNAAnnealedOligonucleotide(0.5pmol/µl)
• 20 µl NegativeControlshRNAAnnealedOligonucleotide(0.5pmol/µl)
• pSIREN-RetroQ-ZsGreenVectorInformationPacket(PT3777-5)
• RetroviralGeneTransferandExpressionUserManual(PT3132-1)
RNAi-Ready pSIREN-RetroQ-DsRed-Express Vector (Cat.No.632487)
• 1 µg RNAi-ReadypSIREN-RetroQ-DsRed-ExpressVector(linearized,25ng/µl)
• 20 µl LuciferaseshRNAAnnealedOligonucleotide(0.5pmol/µl)
• 20 µl NegativeControlshRNAAnnealedOligonucleotide(0.5pmol/µl)
• pSIREN-RetroQ-DsRed-ExpressVectorInformationPacket(PT3830-5)
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• RetroviralGeneTransferandExpressionUserManual(PT3132-1)
RNAi-Ready pSIREN-Shuttle Vector (Cat.No.631527)
• 1 µg RNAi-ReadypSIREN-ShuttleVector(linearized,25ng/µl)
• 20 µl LuciferaseshRNAAnnealedOligonucleotide(0.5pmol/µl)
• 20 µl NegativeControlshRNAAnnealedOligonucleotide(0.5pmol/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)
• 20 µl LuciferaseshRNAAnnealedOligonucleotide(0.5pmol/µl)
• 20 µl NegativeControlshRNAAnnealedOligonucleotide(0.5pmol/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 LuciferaseshRNAAnnealedOligonucleotide(0.5pmol/µl)
• 20 µl NegativeControlshRNAAnnealedOligonucleotide(0.5pmol/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)
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• pSIREN-ShuttleVectorInformationPacket(PT3736-5)
• Adeno-X™ExpressionSystem1UserManual(PT3414-1)
Knockout™ Adenoviral RNAi System 2 (Cat.No.631529)
• 40 µl RNAi-ReadypSIREN-DNRVector(linearized,25ng/µl)
• 20 µl LuciferaseshRNAAnnealedOligonucleotide(0.5pmol/µl)
• 20 µl NegativeControlshRNAAnnealedOligonucleotide(0.5pmol/µl)
• 5x18 µl Adeno-X™LPPromoterlessReactionMixa
• 5 µl CreRecombinase
• 100 µl Adeno-X™LPPromoterlessPrimerMixb
• pSIREN-DNRVectorInformationPacket(PT3738-5)
• Adeno-X™ExpressionSystem1UserManual(PT3414-1)
• 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)
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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)
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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.
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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.
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• 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
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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.
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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
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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.
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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.
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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.
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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).
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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.
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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.
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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.
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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.
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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.
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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.
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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
Protocol No. PT3739-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR63158� 31
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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
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3739-1 3� Version No. PR63158�
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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
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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
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3739-1 3� Version No. PR63158�
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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)
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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)
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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).
Protocol No. PT3739-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR63158� 37
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Notes
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3739-1 38 Version No. PR63158�
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Notes
Protocol No. PT3739-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR63158� 39
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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.
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