慢病毒包装教程Lentivirus Packaging and ProductionWord文档下载推荐.docx

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Plasmid

Description

12260

psPAX2

2ndgenerationpackagingplasmidforproducingviralparticles.psPAX2containsarobustCAGpromoterforefficientexpressionofpackagingproteins.TronolabandAebischerlablentiviralvectorsrequirepsPAX2.ProduceshighertiterthanpCMV-dR8.2dvpr.

12259

pMD2.G

Envelopeplasmidforproducingviralparticles

2ndgenerationsystemdepositedbytheWeinberglab:

8455

pCMV-dR8.2dvpr

2ndgenerationpackagingplasmidforproducingviralparticles

8454

pCMV-VSVG

3rdGenerationPackagingSystem

The3rdgenerationpackagingsystemoffersmaximalbiosafetybutismorecumbersometouse,asitinvolvesthetransfectionoffourdifferentplasmidsintheproducercells（twopackagingplasmids,anenvelopeplasmid,andthelentiviralvector）.

Ifyouwishtousethissystem,youneedtohavealentiviralvectorwithachimeric5'

LTRinwhichtheHIVpromoterisreplacedwithCMVorRSV,thusmakingitTAT-independent.ExamplesofthesevectorsincludepLKO.1,pLL3.7,pLB,pLenti6,pSico,pCL,andpCS.MostAebischerandTronoLablentiviralvectorsCANNOTbeusedwiththissystem.Alentiviralvectorcarryingachimeric5'

LTRcanbepackagedwitheitherthe2ndor3rdgenerationpackagingsystem.

12251

pMDLg/pRRE

3rdgenerationpackagingplasmidforproducingviralparticles

12253

pRSV-Rev

Moreinformation

∙ClickheretobrowseotherRNAivectors,orsearchforplasmidsusingthesearchbaratthetopofthepage.

∙TronoLabwebsiteorLentiweb:

informationandadiscussionforumoncloning,packaging,andotherprotocols.

∙MoffatJet.al.2006.AlentiviralRNAilibraryforhumanandmousegenesappliedtoanarrayedviralhigh-contentscreen.Cell124:

1283-1298.（PubMed）

∙Venturaet.al.2004.Cre-lox-regulatedconditionalRNAinterferencefromtransgenes.PNAS2004Jul13;

101（28）:

10380-5.（PubMed）

∙NaldiniLet.al.1996.Invivogenedeliveryandstabletransductionofnondividingcellsbyalentiviralvector.Science272:

263-267.（PubMed）

∙Dulletal.,AThird-GenerationLentivirusVectorwithaConditionalPackagingSystem.J.Virol.199872（11）:

8463-8472.（PubMed）

∙ZuffereyRet.al.1997.Multiplyattenuatedlentiviralvectorachievesefficientgenedeliveryinvivo.NatBiotechnol15（9）:

871-5.（PubMed）

∙ZuffereyRet.al.1998.Self-inactivatinglentivirusvectorforsafeandefficientinvivogenedelivery.JVirol72（12）:

9873-80.（PubMed）

CellLine

The293TcelllineforproducinglentiviralparticlescanbeobtainedfromGenHunter.

pLKO.1Protocol

pLKO.1-TRCCloningVector

AddgenePlasmid10878.ProtocolVersion1.0.December2006.

CopyrightAddgene2006,AllRightsReserved.Thisprotocolisprovidedforyourconvenience.Seewarrantyinformationinappendix.

Clickhereforaprintablecopy.

TableofContents

∙A.pLKO.1-TRCCloningVector

oA.1TheRNAiConsortium

oA.2MapofpLKO.1

oA.3Relatedplasmids

∙B.DesigningshRNAOligosforpLKO.1

oB.1Determinetheoptimal21-mertargetsinyourgene

oB.2OrderoligoscompatiblewithpLKO.1

∙C.CloningshRNAoligosintopLKO.1

oC.1Recommendedmaterials

oC.2Annealingoligos

oC.3DigestingpLKO.1TRC-CloningVector

oC.4Ligatingandtransformingintobacteria

∙D.ScreeningforInserts

oD.1Recommendedmaterials

oD.2Screeningforinserts

∙E.ProducingLentiviralParticles

oE.1Recommendedmaterials

oE.2Protocolforproducinglentiviralparticles

∙F.InfectingTargetCells

oF.1Recommendedmaterials

oF.2Determiningtheoptimalpuromycinconcentration

oF.3Protocolforlentiviralinfectionandselection

∙G.Safety

∙H.References

oH.1Publishedarticles

oH.2Webresources

∙I.Appendix

oI.1SequenceofpLKO.1TRC-CloningVector

oI.2Recipes

oI.3Warrantyinformation

BacktoTop

A.pLKO.1-TRCCloningVector

A.1TheRNAiConsortium

ThepLKO.1cloningvectoristhebackboneuponwhichTheRNAiConsortium（TRC）hasbuiltalibraryofshRNAsdirectedagainst15,000humanand15,000mousegenes.AddgeneisworkingwiththeTRCtomakethisshRNAcloningvectoravailabletothescientificcommunity.PleaseciteMoffatetal.,Cell2006Mar;

124（6）:

1283-98（PubMed）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.

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'

longterminalrepeat.

RRE

Revresponseelement.

Figure2:

DetailofshRNAinsert.TheU6promoterdirectsRNAPolymeraseIIItranscriptionoftheshRNA.TheshRNAcontains21"

sense"

basesthatareidenticaltothetargetgene,aloopcontaininganXhoIrestrictionsite,and21"

antisense"

basesthatarecomplementarytothe"

bases.TheshRNAisfollowedbyapolyTterminationsequenceforRNAPolymeraseIII.

A.3RelatedProducts

ThefollowingplasmidsavailablefromAddgenearerecommendedforuseinconjunctionwiththepLKO.1TRC-cloningvector.

Plasmid（AddgeneID#）

pLKO.1-TRCcontrol（10879）

Negativecontrolvectorcontainingnon-hairpininsert.

pLKO.1-scrambleshRNA（1864）

NegativecontrolvectorcontainingscrambledshRNA.

psPAX2（12260）

Packagingplasmidforproducingviralparticles.

pMD2.G（12259）

Envelopeplasmidforproducingviralparticles.

Note:

pLKO.1canalsobeusedwithpackagingplasmidpCMV-dR8.2dvpr（Addgene#8455）andenvelopeplasmidpCMV-VSVG（Addgene#8454）fromRobertWeinberg'

slab.Formoreinformation,visitAddgene'

sMammalianRNAiToolspage.

SeveralotherlaboratorieshavedepositedpLKOderivedvectorsthatmayalsobeusefulforyourexperiment.Toseethesevectors,visitAddgene'

swebsiteandsearchfor"

pLKO"

.

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）