pLKO1PURO 慢病毒报装Protocols.docx

pLKO1PURO 慢病毒报装Protocols.docx

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pLKO1PURO慢病毒报装Protocols

Protocols >pLKO.1Protocol

Addgene isaglobal,non-profitplasmidrepositorydedicatedtomakingiteasierforscientiststoshare.

pLKO.1– TRC CloningVector

AddgenePlasmid10878.ProtocolVersion1.0.December2006.

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

TableofContents

∙A.pLKO.1-TRC CloningVector

∙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-TRC CloningVector

A.1TheRNAiConsortium

ThepLKO.1cloningvectoristhebackboneuponwhich TheRNAiConsortium hasbuiltalibraryofshRNAsdirectedagainst15,000humanand15,000mousegenes.Addgeneisworkingwiththe TRC tomakethisshRNAcloningvectoravailabletothescientificcommunity.PleaseciteMoffatetal.,Cell2006Mar;124（6）:

1283-98（'PubMed”:

http:

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

dopt=abstract）inallpublicationsarisingfromtheuseofthisvector.

A.2MapofpLKO.1

pLKO.1isareplication-incompetentlentiviralvectorchosenbythe TRC forexpressionofshRNAs.pLKO.1canbeintroducedintocellsviadirecttransfection,orcanbeconvertedintolentiviralparticlesforsubsequentinfectionofatargetcellline.Onceintroduced,thepuromycinresistancemarkerencodedinpLKO.1allowsforconvenientstableselection.

Figure1:

MapofpLKO.1containinganshRNAinsert.TheoriginalpLKO.1-TRC cloningvectorhasa1.9kbstufferthatisreleasedbydigestionwithAgeIandEcoRI.shRNAoligosareclonedintotheAgeIandEcoRIsitesinplaceofthestuffer.TheAgeIsiteisdestroyedinmostcases（dependingonthetargetsequence）,whiletheEcoRIsiteispreserved.ForacompletemapofpLKO.1containingthe1.9kbstuffer,visit www.addgene.org/10878.

Description

VectorElement

U6

HumanU6promoterdrives RNA Polymerase III transcriptionforgenerationofshRNAtranscripts.

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– TRC control

Negativecontrolvectorcontainingnon-hairpininsert.

pLKO.1–scrambleshRNA

NegativecontrolvectorcontainingscrambledshRNA.

psPAX2

Packagingplasmidforproducingviralparticles.

pMD2.G

Envelopeplasmidforproducingviralparticles.

Note:

pLKO.1canalsobeusedwithpackagingplasmid pCMV-dR8.2dvpr andenvelopeplasmid pCMV-VSVG fromRobertWeinberg’slab.Formoreinformation,visitAddgene’s MammalianRNAiTools page.

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（N 19 ）.Ifnosuitablematchisfound,searchforNAR（N 17 ）YNN,whereNisanynucleotide,Risapurine（A,G）,andYisapyrimidine（C,U）.

∙G-C contentshouldbe36-52%.

∙Sense3’endshouldhavelowstability–atleastoneAorTbetweenposition15-19.

∙Avoidtargetingintrons.

∙Avoidstretchesof4ormorenucleotiderepeats,especiallyrepeatedTsbecausepolyTisaterminationsignalfor RNA polymerase III.

2.Tominimizedegradationofoff-targetmRNAs,useNCBI’s BLAST program.Selectsequencesthathaveatleast3nucleotidemismatchestoallunrelatedgenes.

 Addgenerecommendsthatyouselectmultipletargetsequencesforeachgene.Somesequenceswillbemoreeffectivethanothers.Inaddition,demonstratingthattwodifferentshRNAsthattargetthesamegenecanproducethesamephenotypewillalleviateconcernsaboutoff-targeteffects.

B.2OrderingOligosCompatiblewithpLKO.1

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

Forwardoligo:

5’CCGG—21bpsense—CTCGAG—21bpantisense—TTTTTG 3’

Reverseoligo:

5’AATTCAAAAA—21bpsense—CTCGAG—21bpantisense3’

Forexample,ifthetargetsequenceis（AA）TGCCTACGTTAAGCTATAC,theoligoswouldbe:

Forwardoligo:

5’ CCGG AATGCCTACGTTAAGCTATAC CTCGAG GTATAGCTTAACGTAGGCATT TTTTTG 3’

Reverseoligo:

5’ AATTCAAAAA AATGCCTACGTTAAGCTATAC CTCGAG GTATAGCTTAACGTAGGCATT 3’

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C.CloningOligosintopLKO.1

ThepLKO.1-TRC cloningvectorcontainsa1.9kbstufferthatisreleasedupondigestionwithEcoRIandAgeI.

TheoligosfromsectionBcontaintheshRNAsequenceflankedbysequencesthatarecompatiblewiththestickyendsofEcoRIandAgeI.ForwardandreverseoligosareannealedandligatedintothepLKO.1vector,producingafinalplasmidthatexpressestheshRNAofinterest.

C.1RecommendedMaterials

Material

Vendorandcatalog#

AgeI

NewEnglandBiolabs（NEB）#R0552S

EcoRI

NEB #R0101S

T4 DNA ligase

NEB #M0202S

NEB buffer2

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

5μL10x NEB buffer2

35μLddH2O

2.Incubatefor4minutesat95°Cina PCR machineorinabeakerofboilingwater.

3.Ifusinga PCR machine,incubatethesampleat70°Cfor10minutesthenslowlycooltoroomtemperatureovertheperiodofseveralhours.Ifusingabeakerofwater,removethebeakerfromtheflame,andallowthewatertocooltoroomtemperature.Thiswilltakeafewhours,butitisimportantforthecoolingtooccurslowlyfortheoligostoanneal.

C.3DigestingpLKO.1 TRC CloningVector

1.DigestpLKO.1TRC-cloningvectorwithAgeI.Mix:

6μgpLKO.1TRC-cloningvector（maxipreporminiprep DNA）

5μL10x NEB buffer1

1μLAgeI

to50μLddH2O

>Incubateat37°Cfor2hours.

2.PurifywithQiaquickgelextractionkit.Elutein30μLofddH2O.

3.DigesteluatewithEcoRI.Mix:

30μLpLKO.1TRC-cloningvectordigestedwithAgeI

5μL10x NEB bufferforEcoRI

1μLEcoRI

14μLddH2O

>Incubateat37°Cfor2hours.

4.Rundigested DNA on0.8%lowmeltingpointagarosegeluntilyoucandistinctlysee2bands,one7kbandone1.9kb.Cutoutthe7kbbandandplaceinasterilemicrocentrifugetube.

 Whenvisualizing DNA fragmentstobeusedforligation,useonlylong-wavelengthUVlight.ShortwavelengthUVlightwillincreasethechanceofdamagingthe DNA.

5.Purifythe DNA usingaQiaquickgelextractionkit.Elutein30μLofddH2O.

6.Measurethe DNA concentration.

C.4LigatingandTransformingintoBacteria

1.Useyourligationmethodofchoice.ForastandardT4ligation,mix:

2μLannealedoligofromstepC.2.

20ngdigestedpLKO.1TRC-cloningvectorfromstepC.3.（Ifyouwereunabletomeasurethe DNA concentration,use1μL）

2μL10x NEB T4 DNA ligasebuffer

1μLNEB T4 DNA ligase

to20μLddH2O

>Incubateat16°Cfor4-20hours.

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

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D.ScreeningforInserts

Youmayscreenforplasmidsthatweresuccessfullyligatedbyrestrictionenzymedigestion.However,onceyouhaveidentifiedthepositiveclones,itis