shRNA载体质粒plko1mannual.docx

shRNA载体质粒plko1mannual.docx

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shRNA载体质粒plko1mannual

pLKO.1–TRCCloningVector

AddgenePlasmid10878.ProtocolVersion1.0.December2006.

CopyrightAddgene2006,AllRightsReserved.Thisprotocolisprovidedforyourconvenience.See“warrantyinformation”inappendix.

TableofContents

∙A.pLKO.1-TRCCloningVector

∙A.1TheRNAiConsortium

∙A.2MapofpLKO.1

∙A.3Relatedplasmids

∙B.DesigningshRNAOligosforpLKO.1

∙B.1Determinetheoptimal21-mertargetsinyourgene

∙B.2OrderoligoscompatiblewithpLKO.1

∙C.CloningshRNAoligosintopLKO.1

∙C.1Recommendedmaterials

∙C.2Annealingoligos

∙C.3DigestingpLKO.1TRC-CloningVector

∙C.4Ligatingandtransformingintobacteria

∙D.ScreeningforInserts

∙D.1Recommendedmaterials

∙D.2Screeningforinserts

∙E.ProducingLentiviralParticles

∙E.1Recommendedmaterials

∙E.2Protocolforproducinglentiviralparticles

∙F.InfectingTargetCells

∙F.1Recommendedmaterials

∙F.2Determiningtheoptimalpuromycinconcentration

∙F.3Protocolforlentiviralinfectionandselection

∙G.Safety

∙H.References

∙H.1Publishedarticles

∙H.2Webresources

∙I.Appendix

∙I.1SequenceofpLKO.1TRC-CloningVector

∙I.2Recipes

∙I.3Warrantyinformation

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A.pLKO.1-TRCCloningVector

A.1TheRNAiConsortium

ThepLKO.1cloningvectoristhebackboneuponwhichTheRNAiConsortiumhasbuiltalibraryofshRNAsdirectedagainst15,000humanand15,000mousegenes.AddgeneisworkingwiththeTRCtomakethisshRNAcloningvectoravailabletothescientificcommunity.PleaseciteMoffatetal.,Cell2006Mar;124（6）:

1283-98（'PubMed”:

http:

//www.ncbi.nlm.nih.gov/pubmed/16564017?

dopt=abstract）inallpublicationsarisingfromtheuseofthisvector.

A.2MapofpLKO.1

pLKO.1isareplication-incompetentlentiviralvectorchosenbytheTRCforexpressionofshRNAs.pLKO.1canbeintroducedintocellsviadirecttransfection,orcanbeconvertedintolentiviralparticlesforsubsequentinfectionofatargetcellline.Onceintroduced,thepuromycinresistancemarkerencodedinpLKO.1allowsforconvenientstableselection.

Figure1:

MapofpLKO.1containinganshRNAinsert.TheoriginalpLKO.1-TRCcloningvectorhasa1.9kbstufferthatisreleasedbydigestionwithAgeIandEcoRI.shRNAoligosareclonedintotheAgeIandEcoRIsitesinplaceofthestuffer.TheAgeIsiteisdestroyedinmostcases（dependingonthetargetsequence）,whiletheEcoRIsiteispreserved.ForacompletemapofpLKO.1containingthe1.9kbstuffer,visitwww.addgene.org/10878.

Description

VectorElement

U6

HumanU6promoterdrivesRNAPolymeraseIIItranscriptionforgenerationofshRNAtranscripts.

cPPT

Centralpolypurinetract,cPPT,improvestransductionefficiencybyfacilitatingnuclearimportofthevector’spreintegrationcomplexinthetransducedcells.

hPGK

Humanphosphoglyceratekinasepromoterdrivesexpressionofpuromycin.

PuroR

PuromycinresistancegeneforselectionofpLKO.1plasmidinmammaliancells.

sin3’LTR

3’Self-inactivatinglongterminalrepeat.

f1ori

f1bacterialoriginofreplication.

AmpR

AmpicillinresistancegeneforselectionofpLKO.1plasmidinbacterialcells

pUCori

pUCbacterialoriginofreplication.

5’LTR

5’longterminalrepeat.

RRE

Revresponseelement.

A.3RelatedProducts

ThefollowingplasmidsavailablefromAddgenearerecommendedforuseinconjunctionwiththepLKO.1TRC-cloningvector.

Plasmid（AddgeneID#）

Description

pLKO.1–TRCcontrol

Negativecontrolvectorcontainingnon-hairpininsert.

pLKO.1–scrambleshRNA

NegativecontrolvectorcontainingscrambledshRNA.

psPAX2

Packagingplasmidforproducingviralparticles.

pMD2.G

Envelopeplasmidforproducingviralparticles.

Note:

pLKO.1canalsobeusedwithpackagingplasmidpCMV-dR8.2dvprandenvelopeplasmidpCMV-VSVGfromRobertWeinberg’slab.Formoreinformation,visitAddgene’sMammalianRNAiToolspage.

SeveralotherlaboratorieshavedepositedpLKOderivedvectorsthatmayalsobeusefulforyourexperiment.Toseethesevectors,visitAddgene’swebsiteand“searchfor“pLKO”“.

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B.DesigningshRNAOligosforpLKO.1

B.1DeterminingtheOptimal21-merTargetsinyourGene

Selectionofsuitable21-mertargetsinyourgeneisthefirststeptowardefficientgenesilencing.Methodsfortargetselectionarecontinuouslybeingimproved.Belowaresuggestionsfortargetselection.

1.UseansiRNAselectiontooltodetermineasetoftop-scoringtargetsforyourgene.Forexample,theWhiteheadInstituteforBiomedicalResearchhostsansiRNASelectionProgramthatcanbeaccessedafterafreeregistration（http:

//jura.wi.mit.edu/bioc/siRNAext/）.IfyouhaveMacOSX,anotherexcellentprogramisiRNAi,whichisprovidedfreebythecompanyMekentosj（

AsummaryofguidelinesfordesigningsiRNAswitheffectivegenesilencingisincludedhere:

∙Startingat25ntdownstreamofthestartcodon（ATG）,searchfor21ntsequencesthatmatchthepatternAA（N19）.Ifnosuitablematchisfound,searchforNAR（N17）YNN,whereNisanynucleotide,Risapurine（A,G）,andYisapyrimidine（C,U）.

∙G-Ccontentshouldbe36-52%.

∙Sense3’endshouldhavelowstability–atleastoneAorTbetweenposition15-19.

∙Avoidtargetingintrons.

∙Avoidstretchesof4ormorenucleotiderepeats,especiallyrepeatedTsbecausepolyTisaterminationsignalforRNApolymeraseIII.

2.Tominimizedegradationofoff-targetmRNAs,useNCBI’sBLASTprogram.Selectsequencesthathaveatleast3nucleotidemismatchestoallunrelatedgenes.

Addgenerecommendsthatyouselectmultipletargetsequencesforeachgene.Somesequenceswillbemoreeffectivethanothers.Inaddition,demonstratingthattwodifferentshRNAsthattargetthesamegenecanproducethesamephenotypewillalleviateconcernsaboutoff-targeteffects.

B.2OrderingOligosCompatiblewithpLKO.1

TogenerateoligosforcloningintopLKO.1,insertyoursenseandantisensesequencesfromstepB.1intotheoligosbelow.Donotchangetheends;thesebasesareimportantforcloningtheoligosintothepLKO.1TRC-cloningvector.

Forwardoligo:

5’CCGGT—21bpsense—CTCGAG—21bpantisense—TTTTTG3’

Reverseoligo:

5’AATTCAAAAA—21bpsense—CTCGAG—21bpantisense—A’

Forexample,ifthetargetsequenceis（AA）TGCCTACGTTAAGCTATAC,theoligoswouldbe:

Forwardoligo:

5’CCGGAATGCCTACGTTAAGCTATACCTCGAGGTATAGCTTAACGTAGGCATTTTTTTG3’

Reverseoligo:

5’AATTCAAAAAAATGCCTACGTTAAGCTATACCTCGAGGTATAGCTTAACGTAGGCATT3’

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C.CloningOligosintopLKO.1

ThepLKO.1-TRCcloningvectorcontainsa1.9kbstufferthatisreleasedupondigestionwithEcoRIandAgeI.

TheoligosfromsectionBcontaintheshRNAsequenceflankedbysequencesthatarecompatiblewiththestickyendsofEcoRIandAgeI.ForwardandreverseoligosareannealedandligatedintothepLKO.1vector,producingafinalplasmidthatexpressestheshRNAofinterest.

C.1RecommendedMaterials

Material

Vendorandcatalog#

AgeI

NewEnglandBiolabs（NEB）#R0552S

EcoRI

NEB#R0101S

T4DNAligase

NEB#M0202S

NEBbuffer2

NEB#B7002S

DH5alphacompetentcells

Invitrogen#18258-012

Qiaquickgelextractionkit

Qiagen#28704

Lowmeltingpointagarose

Sigma#A9414

LuriaBrothAgar（LBagar）

AmericanBioanalytical:

#AB01200-02000

Ampicillin

VWR:

#7177-48-2.Useat100μg/mL.

Carbenicillin

VWR:

#80030-956.Useat100μg/mL.

C.2AnnealingOligos

1.ResuspendoligosinddH2Otoaconcentrationof20μM,thenmix:

5μLForwardoligo

5μLReverseoligo

5μL10xNEBbuffer2

35μLddH2O

2.Incubatefor4minutesat95°CinaPCRmachineorinabeakerofboilingwater.

3.IfusingaPCRmachine,incubatethesampleat70°Cfor10minutesthenslowlycooltoroomtemperatureovertheperiodofseveralhours.Ifusingabeakerofwater,removethebeakerfromtheflame,andallowthewatertocooltoroomtemperature.Thiswilltakeafewhours,butitisimportantforthecoolingtooccurslowlyfortheoligostoanneal.

C.3DigestingpLKO.1TRCCloningVector

1.DigestpLKO.1TRC-cloningvectorwithAgeI.Mix:

6μgpLKO.1TRC-cloningvector（maxipreporminiprepDNA）

5μL10xNEBbuffer1

1μLAgeI

to50μLddH2O

>Incubateat37°Cfor2hours.

2.PurifywithQiaquickgelextractionkit.Elutein30μLofddH2O.

3.DigesteluatewithEcoRI.Mix:

30μLpLKO.1TRC-cloningvectordigestedwithAgeI

5μL10xNEBbufferforEcoRI

1μLEcoRI

14μLddH2O

>Incubateat37°Cfor2hours.

4.RundigestedDNAon0.8%lowmeltingpointagarosegeluntilyoucandistinctlysee2bands,one7kbandone1.9kb.Cutoutthe7kbbandandplaceinasterilemicrocentrifugetube.

WhenvisualizingDNAfragmentstobeusedforligation,useonlylong-wavelengthUVlight.ShortwavelengthUVlightwillincreasethechanceofdamagingtheDNA.

5.PurifytheDNAusingaQiaquickgelextractionkit.Elutein30μLofddH2O.

6.MeasuretheDNAconcentration.

C.4LigatingandTransformingintoBacteria

1.Useyourligationmethodofchoice.ForastandardT4ligation,mix:

2μLannealedoligofromstepC.2.

20ngdigestedpLKO.1TRC-cloningvectorfromstepC.3.（IfyouwereunabletomeasuretheDNAconcentration,use1μL）

2μL10xNEBT4DNAligasebuffer

1μLNEBT4DNAligase

to20μLddH2O

>Incubateat16°Cfor4-20hours.

2.Transform2μLofligationmixinto25μLcompetentDH5alphacells,followingmanufacturer’sprotocol.PlateonLBagarplatescontaining100μg/mLampicillinorcarbenicillin（anampicillinanalog）.

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D.ScreeningforInserts

Youmayscreenforplasmidsthatweresuccessfullyligatedbyrestrictionenzymedigestion.However,onceyouhaveidentifiedthepositiveclones,