慢病毒包装教程Lentivirus Packaging and Production.docx

慢病毒包装教程Lentivirus Packaging and Production.docx

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慢病毒包装教程LentivirusPackagingandProduction

LentivirusPackagingandProduction

ThelaboratoriesofDidierTrono（EPFL）andRobertWeinberg（WhiteheadInstitute）havedepositedplasmidsfortheproductionoflentiviralparticles.Theseplasmidscanbeusedwithmanylentiviralvectors,includingTheRNAiConsortiumshRNAvectorsbeingdistributedbySigma（i.e.MISSIONshRNAs）andOpenBiosystems（i.e.TRCshRNAs）.

Overview

Forproducinglentiviralparticles,youtypicallyneedthreecomponents:

1）alentiviralvector,suchaspLKO.1orpLVTHM,containingtheshRNAortransgene,2）apackagingvector,suchaspsPAX2orpCMV-dR8.2dvpr,and3）anenvelopevector,suchaspMD2.GorpCMV-VSVG.

Formostapplications,youcanproduceviralparticlesbytransienttransfectionof293Tcellswitha2ndgenerationpackagingsystem（e.g.packagingplasmidpsPAX2andenvelopeplasmidpMD2.G）.

2ndGenerationPackagingSystem

Ingeneral,lentiviralvectorswithawildtype5'LTRneedthe2ndgenerationpackagingsystembecausethesevectorsrequireTATforactivation.AlllentiviralvectorsfromtheTronoorAebischerlabrequirepackagingwitha2ndgenerationsystem.

Belowaretwo2ndgenerationsystems.LentiviralplasmidsbasedonpLKO.1canbepackagedwitheithersystem,althoughthefirstsystemhasbeenreportedtoproducehighertiter.SeeAddgene'spLKO.1Protocolforproducinglentiviralparticles.

2ndgenerationsystemdepositedbytheTronolab:

ID

Plasmid

Description

12260

psPAX2

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

12259

pMD2.G

Envelopeplasmidforproducingviralparticles

2ndgenerationsystemdepositedbytheWeinberglab:

ID

Plasmid

Description

8455

pCMV-dR8.2dvpr

2ndgenerationpackagingplasmidforproducingviralparticles

8454

pCMV-VSVG

Envelopeplasmidforproducingviralparticles

3rdGenerationPackagingSystem

The3rdgenerationpackagingsystemoffersmaximalbiosafetybutismorecumbersometouse,asitinvolvesthetransfectionoffourdifferentplasmidsintheproducercells（twopackagingplasmids,anenvelopeplasmid,andthelentiviralvector）.

Ifyouwishtousethissystem,youneedtohavealentiviralvectorwithachimeric5'LTRinwhichtheHIVpromoterisreplacedwithCMVorRSV,thusmakingitTAT-independent.ExamplesofthesevectorsincludepLKO.1,pLL3.7,pLB,pLenti6,pSico,pCL,andpCS.MostAebischerandTronoLablentiviralvectorsCANNOTbeusedwiththissystem.Alentiviralvectorcarryingachimeric5'LTRcanbepackagedwitheitherthe2ndor3rdgenerationpackagingsystem.

ID

Plasmid

Description

12251

pMDLg/pRRE

3rdgenerationpackagingplasmidforproducingviralparticles

12253

pRSV-Rev

3rdgenerationpackagingplasmidforproducingviralparticles

12259

pMD2.G

Envelopeplasmidforproducingviralparticles

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.2Annealing