pLKO.1.PURO-慢病毒报装Protocols

ProtocolspLKO.1ProtocolAddgeneisaglobal,non-profitplasmidrepositorydedicatedtomakingiteasierforscientiststoshare.pLKO.1–TRCCloningVectorAddgenePlasmid10878.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.3WarrantyinformationBacktoTopA.pLKO.1-TRCCloningVectorA.1TheRNAiConsortiumThepLKO.1cloningvectoristhebackboneuponwhichTheRNAiConsortiumhasbuiltalibraryofshRNAsdirectedagainst15,000humanand15,000mousegenes.AddgeneisworkingwiththeTRCtomakethisshRNAcloningvectoravailabletothescientificcommunity.PleaseciteMoffatetal.,Cell2006Mar;124(6):1283-98('PubMed”:=abstract)inallpublicationsarisingfromtheuseofthisvector.A.2MapofpLKO.1pLKO.1isareplication-incompetentlentiviralvectorchosenbytheTRCforexpressionofshRNAs.pLKO.1canbeintroducedintocellsviadirecttransfection,orcanbeconvertedintolentiviralparticlesforsubsequentinfectionofatargetcellline.Onceintroduced,thepuromycinresistancemarkerencodedinpLKO.1allowsforconvenientstableselection.Figure1:MapofpLKO.1containinganshRNAinsert.TheoriginalpLKO.1-TRCcloningvectorhasa1.9kbstufferthatisreleasedbydigestionwithAgeIandEcoRI.shRNAoligosareclonedintotheAgeIandEcoRIsitesinplaceofthestuffer.TheAgeIsiteisdestroyedinmostcases(dependingonthetargetsequence),whiletheEcoRIsiteispreserved.ForacompletemapofpLKO.1containingthe1.9kbstuffer,visit’spreintegrationcomplexinthetransducedcells.hPGKHumanphosphoglyceratekinasepromoterdrivesexpressionofpuromycin.PuroRPuromycinresistancegeneforselectionofpLKO.1plasmidinmammaliancells.sin3’LTR3’Self-inactivatinglongterminalrepeat.f1orif1bacterialoriginofreplication.AmpRAmpicillinresistancegeneforselectionofpLKO.1plasmidinbacterialcellspUCoripUCbacterialoriginofreplication.5’LTR5’longterminalrepeat.RRERevresponseelement.A.3RelatedProductsThefollowingplasmidsavailablefromAddgenearerecommendedforuseinconjunctionwiththepLKO.1TRC-cloningvector.Plasmid(AddgeneID#)DescriptionpLKO.1–TRCcontrolNegativecontrolvectorcontainingnon-hairpininsert.pLKO.1–scrambleshRNANegativecontrolvectorcontainingscrambledshRNA.psPAX2Packagingplasmidforproducingviralparticles.pMD2.GEnvelopeplasmidforproducingviralparticles.Note:pLKO.1canalsobeusedwithpackagingplasmidpCMV-dR8.2dvprandenvelopeplasmidpCMV-VSVGfromRobertWeinberg’slab.Formoreinformation,visitAddgene’sMammalianRNAiToolspage.SeveralotherlaboratorieshavedepositedpLKOderivedvectorsthatmayalsobeusefulforyourexperiment.Toseethesevectors,visitAddgene’swebsiteand“searchfor“pLKO”“.BacktoTopB.DesigningshRNAOligosforpLKO.1B.1DeterminingtheOptimal21-merTargetsinyourGeneSelectionofsuitable21-mertargetsinyourgeneisthefirststeptowardefficientgenesilencing.Methodsfortargetselectionarecontinuouslybeingimproved.Belowaresuggestionsfortargetselection.1.UseansiRNAselectiontooltodetermineasetoftop-scoringtargetsforyourgene.Forexample,theWhiteheadInstituteforBiomedicalResearchhostsansiRNASelectionProgramthatcanbeaccessedafterafreeregistration().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,demonstratingthattwodifferentshRNAsthattargetthesameg