慢病毒包装教程.docx
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慢病毒包装教程.docx
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慢病毒包装教程
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.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.
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.
Figure2:
DetailofshRNAinsert.TheU6promoterdirectsRNAPolymeraseIIItranscriptionoftheshRNA.TheshRNAcontains21"sense"basesthatareidenticaltothetargetgene,aloopcontaininganXhoIrestrictionsite,and21"antisense"basesthatarecomplementarytothe"sense"bases.TheshRNAisfollowedbyapolyTterminationsequenceforRNAPolymeraseIII.
A.3RelatedProducts
ThefollowingplasmidsavailablefromAddgenearerecommendedforuseinconjunctionwiththepLKO.1TRC-cloningvector.
Plasmid(AddgeneID#)
Description
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".
BacktoTop
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,Ri
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