7QSP )FMQFS'SFF 4ZTUFN - Takara Bio · Viruses produced with AAV-based vectors could, depending on...
Transcript of 7QSP )FMQFS'SFF 4ZTUFN - Takara Bio · Viruses produced with AAV-based vectors could, depending on...
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Cat. # 6230, 6650 - 6657,6668, 6669, 6673
Product Manual
AAVpro® Helper Free System
For Research Use
v201611Da
【 For China/Korea/India/Europe 】
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Table of Contents
I. Description........................................................................................................ 4
II. Components.................................................................................................... 7
III. Storage.............................................................................................................13
IV. MaterialsRequiredbutnotProvided...................................................13
V. OverviewofAAVParticlePreparation..................................................14
VI. Protocol............................................................................................................14
VII. MeasurementofVirusTiter......................................................................17
VIII. ReferenceData..............................................................................................18
IX. References.......................................................................................................22
X. RelatedProducts..........................................................................................22
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Safety & Handling of Adeno-Associated Virus VectorsTheprotocolsinthisusermanualrequirethehandlingofadeno-associatedvirusvectors.Itisimperativetofullyunderstandthepotentialhazardsofandnecessaryprecautionsforlaboratoryuseofthesevectors.VirusesproducedwithAAV-basedvectorscould,dependingonyourgeneinsert,bepotentiallyhazardous.Similarvectorshavebeenapprovedforhumangenetherapytrials,attestingtotheirpotentialabilitytoexpressgenesinvivo.Forthesereasons,duecautionmustbeexercisedintheproductionandhandlingofanyrecombinantviruses.FollowallapplicableguidelinesforresearchinvolvingrecombinantDNA.Takeappropriatesafetymeasureswhenproducingorhandlingrecombinantadeno-associatedviruses,includingworkinginabiologicalsafetycabinetandwearingprotectivelaboratorycoats,faceprotection,andgloves.
Available AAVpro ProductspAAV-ZsGreen1Vector Cat.#6231AAVpro®PurificationKit(AllSerotypes) Cat.#6666AAVpro®PurificationKit(AAV2) Cat.#6232AAVpro®TitrationKit(forRealTimePCR)Ver.2 Cat.#6233AAVpro®ExtractionSolution Cat.#6235AAVpro®PackagingPlasmid(AAV1) Cat.#6672AAVpro®PackagingPlasmid(AAV2) Cat.#6234AAVpro®PackagingPlasmid(AAV5) Cat.#6664AAVpro®PackagingPlasmid(AAV6) Cat.#6665AAVpro®293TCellLine Cat.#632273
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I. DescriptionI-1. Adeno-Associated VirusAdeno-AssociatedVirus(AAV)isanon-envelopedvirusthatbelongstotheParvovirusfamilyoftheDependovirusgenus.AAVisnotthoughttobepathogenictohumansandonlyreplicatesinthepresenceofahelpervirus,suchasadenovirusorherpesvirus.TheAAVgenomeisalinear,single-strandDNAmoleculeofapproximately4.7kbthathasterminalhairpinstructurescalledinvertedterminalrepeats(ITRs)atbothends.ITRsfunctionasoriginsofgenomicreplicationandcontributetopackagingofviralparticles.TheAAVgenomeincludesthreeopenreadingframes(Figure1):Rep,whichencodesaproteininvolvedinreplicationandtranscription;Cap,whichencodescapsidproteins;andAAP,whichencodesanon-structuralproteinnecessaryforformationofviralparticles.TheRepregioncodesfor4differentproteins(Rep78,Rep68,Rep52,andRep40),andtheCapregioncodesfor3differentproteins(VP1,VP2,andVP3).
Therearemorethan100serotypesofAAV,andthehostspecificityandcharacteristicsofthevirusdifferamongserotypes.TaKaRaBioInc.provideskitsforpreparationofAAVserotype1(AAV1),serotype2(AAV2),serotype5(AAV5),andserotype6(AAV6).AAVserotype2(AAV2)isthemostcommonlyusedserotypeinAAV-basedresearch,includinggenetherapy,andischaracterizedbyabroadhostrange.ThetissuehostrangeofAAV1,AAV5,andAAV6arehighlylimited.AAV1hashightransductionefficiencyformuscle,liver,respiratorytract,andcentralnervoussystem.AAV5hashightransductionefficiencyforcentralnervoussystem,liver,andretina.AAV6hashightransductionefficiencyforcardiomyocyte,muscle,andliver.
Adeno-associatedvirusvectors(AAVvectors)exploitthepropertiesofAAVfortransductionofgenestocellsandorganisms.AAVvectorsareusedasresearchtoolsandalsoasvectorsforgenetherapy.Inaddition,AAVvectorsaregenerallyconsideredsaferthanadenoviralandretroviralvectors.AAVvectorscanbeusedtotransducegenesintobothproliferatingandnon-proliferatingcellsandcanimpartlong-termexpressioninnon-dividingcells.Inaddition,AAVhaslittleimmunogenicityandissuitableforthetransductionofgenesintoanimals(asaninvivotransductiontool).
Figure1. AAVgenomestructureandopenreadingframes.
Rep
ITR
Cap
Rep78
Rep68
Rep52
Rep40
VP1
VP2
VP3
AAP
ITR
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l-2. PrinciplesEachAAVproHelperFreeSystemenablesthepreparationofAAVparticlesofserotype1,2,5,or6withouttheuseofahelpervirus.TheAAVparticlesproducedcanbeusedtoobtaintransientexpressionofthetargetgeneinmammaliancellsorindividualanimals.Forinvivoapplications,purificationofAAVvectorwiththeAAVproPurificationKit(AllSerotypes)(Cat.#6666)isrecommended.ForAAV2,AAVproPurificationKit(AAV2)(Cat.#6232)isalsoavailable.
l-3. FeaturesA. Preparation of AAV Vector with the AAV Helper-Free SystemTheAAVHelperFreeSystemsareuniquesystemsforthepreparationofhigh-titerAAVparticleswithouttheuseofahelpervirus(Figure2).EachkitincludesthreeplasmidsencodingthefactorsnecessarytopreparerecombinantAAVparticlesbytransfectionintoHEK293cells.
•pAAVVector:PlasmidcontainingapromoterforgeneexpressionandtwoITRs pAAV-CMVVector(Cat.#6673,6230,6650,6651)
Plasmidcontainsasiteforcloningageneofinterest(GOI).TheGOIisexpressedfromaCMVpromoter.ThesizeoftheGOIclonedintothepAAV-CMVvectorshouldbe<2.5kbasthereisalimittothesizeofDNAthatcanbeencapsulatedinAAVparticles.
pAAV-CRERecombinaseVector(Cat.#6668,6652,6653,6654)ThisvectorisusedtoprepareAAVparticlesthatwilldelivertheloxP-dependentCrerecombinasegene.Crerecombinasehasbeenwidelyusedforgeneratingtransgenicmiceandforvariousscreeningassays.
pAAV-LacZVector(Cat.#6669,6655,6656,6657)ThisvectorisusedtoprepareAAVparticlesthatwilldeliveraLacZexpressioncassette.AAV-LacZparticlescanbeusedasacontrolforinvitroandinvivogenetransfer.
•pRCVector:PlasmidthatexpressestheRepgeneofAAV2andtheCapgeneofeachserotypebelow.pRC1Vector: Serotype1(Cat.#6673,6668,6669)pRC2-mi342Vector*: Serotype2(Cat.#6230,6652,6655)pRC5Vector: Serotype5(Cat.#6650,6653,6656)pRC6Vector: Serotype6(Cat.#6651,6654,6657)
•pHelperVector:PlasmidthatexpressesadenovirusE2A,E4,andVA
* pRC2-mi342expresseshsa-miR-342,ahumanmicroRNAthatincreasesAAV2titerinvectorpreparationsystems.Itincreasestiterbyapproximately2-foldascomparedtoordinarypRC2vectorsthatexpressonlyRepandCap(VIII.ReferenceData).
AAVpro® Helper Free System
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B. AAV Particle Extraction using AAV Extraction SolutionExtractionofAAVparticlesfromAAV-producingcellsisconventionallyperformedbyfreeze-thaworsonicationmethods;however,thesemethodsaretimeconsumingandrequirespecialequipment.ThiskitincludesAAVExtractionSolutionsthatallowsimpleandefficientAAVparticleisolationwhileminimizingproteinandnucleicacidcontamination.TheAAVproExtractionSolution(Cat.#6235),whichcontainsAAVproExtractionSolutionsAandB,canbepurchasedseparately.
Figure2. PreparationofAAVparticlesusingtheAAVproHelperFreeSystem.
pAAVVectorpRCVectorpHelperVector
Transfection
293CellAAVssDNA
Replication
TranscriptionandTranslation
AAVExtractionSolution
AAVParticles
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II. ComponentsEachkitincludesthreeplasmidsencodingthefactorsnecessarytopreparerecombinantAAVparticles,andreagentsforextractingAAVparticlesfromproducercells.
[Serotype 1 ]AAVproHelperFreeSystem(AAV1)(Cat.#6673)1. pAAV-CMVVector(1μg/μl) 20μl2. pRC1Vector(1μg/μl) 20μl3. pHelperVector(1μg/μl) 20μl4. AAVExtractionSolutionA 1.5mlx35. AAVExtractionSolutionB 150μlx3
AAVproHelperFreeSystem(AAV1-CRERecombinase)(Cat.#6668)1. pAAV-CRERecombinaseVector(1μg/μl) 20μl2. pRC1Vector(1μg/μl) 20μl3. pHelperVector(1μg/μl) 20μl4. AAVExtractionSolutionA 1.5mlx35. AAVExtractionSolutionB 150μlx3
AAVproHelperFreeSystem(AAV1-LacZ)(Cat.#6669)1. pAAV-LacZVector(1μg/μl) 20μl2. pRC1Vector(1μg/μl) 20μl3. pHelperVector(1μg/μl) 20μl4. AAVExtractionSolutionA 1.5mlx35. AAVExtractionSolutionB 150μlx3
[Serotype 2 ]AAVproHelperFreeSystem(AAV2)(Cat.#6230)1. pAAV-CMVVector(1μg/μl) 20μl2. pRC2-mi342Vector(1μg/μl) 20μl3. pHelperVector(1μg/μl) 20μl4. AAVExtractionSolutionA 1.5mlx35. AAVExtractionSolutionB 150μlx3
AAVproHelperFreeSystem(AAV2-CRERecombinase)(Cat.#6652)1. pAAV-CRERecombinaseVector(1μg/μl) 20μl2. pRC2-mi342Vector(1μg/μl) 20μl3. pHelperVector(1μg/μl) 20μl4. AAVExtractionSolutionA 1.5mlx35. AAVExtractionSolutionB 150μlx3
AAVproHelperFreeSystem(AAV2-LacZ)(Cat.#6655)1. pAAV-LacZVector(1μg/μl) 20μl2. pRC2-mi342Vector(1μg/μl) 20μl3. pHelperVector(1μg/μl) 20μl4. AAVExtractionSolutionA 1.5mlx35. AAVExtractionSolutionB 150μlx3
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[Serotype 5 ]AAVproHelperFreeSystem(AAV5)(Cat.#6650)1. pAAV-CMVVector(1μg/μl) 20μl2. pRC5Vector(1μg/μl) 20μl3. pHelperVector(1μg/μl) 20μl4. AAVExtractionSolutionA 1.5mlx35. AAVExtractionSolutionB 150μlx3
AAVproHelperFreeSystem(AAV5-CRERecombinase)(Cat.#6653)1. pAAV-CRERecombinaseVector(1μg/μl) 20μl2. pRC5Vector(1μg/μl) 20μl3. pHelperVector(1μg/μl) 20μl4. AAVExtractionSolutionA 1.5mlx35. AAVExtractionSolutionB 150μlx3
AAVproHelperFreeSystem(AAV5-LacZ)(Cat.#6656)1. pAAV-LacZVector(1μg/μl) 20μl2. pRC5Vector(1μg/μl) 20μl3. pHelperVector(1μg/μl) 20μl4. AAVExtractionSolutionA 1.5mlx35. AAVExtractionSolutionB 150μlx3
[Serotype 6 ]AAVproHelperFreeSystem(AAV6)(Cat.#6651)1. pAAV-CMVVector(1μg/μl) 20μl2. pRC6Vector(1μg/μl) 20μl3. pHelperVector(1μg/μl) 20μl4. AAVExtractionSolutionA 1.5mlx35. AAVExtractionSolutionB 150μlx3
AAVproHelperFreeSystem(AAV6-CRERecombinase)(Cat.#6654)1. pAAV-CRERecombinaseVector(1μg/μl) 20μl2. pRC6Vector(1μg/μl) 20μl3. pHelperVector(1μg/μl) 20μl4. AAVExtractionSolutionA 1.5mlx35. AAVExtractionSolutionB 150μlx3
AAVproHelperFreeSystem(AAV6-LacZ)(Cat.#6657)1. pAAV-LacZVector(1μg/μl) 20μl2. pRC6Vector(1μg/μl) 20μl3. pHelperVector(1μg/μl) 20μl4. AAVExtractionSolutionA 1.5mlx35. AAVExtractionSolutionB 150μlx3
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Highvolumesetsconsistingofvirus-productionplasmids(components2and3)canbepurchasedseparately.
AAVproPackagingPlasmid(AAV1)(Cat.#6672)1.pRC1Vector(1μg/μl) 0.5mlx22.pHelperVector(1μg/μl) 0.5mlx2
AAVproPackagingPlasmid(AAV2)(Cat.#6234)1.pRC2-mi342Vector(1μg/μl) 0.5mlx22.pHelperVector(1μg/μl) 0.5mlx2
AAVproPackagingPlasmid(AAV5)(Cat.#6664)1.pRC5Vector(1μg/μl) 0.5mlx22.pHelperVector(1μg/μl) 0.5mlx2
AAVproPackagingPlasmid(AAV6)(Cat.#6665)1.pRC6Vector(1μg/μl) 0.5mlx22.pHelperVector(1μg/μl) 0.5mlx2
AAVpro® Helper Free System
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Ampr
pUCori
ITR
ITR
MCS
CMV promoter
β-globinintron
hGHpolyA
pAAV-CMV Vector(5,031 bp)
EcoR VHind III
EcoR VAfl IISfi I
Figure3. pAAV-CMVvectormapandmultiplecloningsite(MCS).
1471 GAATTGGGATTCGCGAGAATTCTCTAGAGTCGACACTAGTGCGGATCCAC 1520 CTTAACCCTAAGCGCTCTTAAGAGATCTCAGCTGTGATCACGCCTAGGTG
EcoR I
Xba INru I Spe I
Sal I Acc IHinc I BamH I
β-globinintron
MCS
Ampr
pUCori
ITR
ITR
CMVpromoter
Humanβ-globinintron
Cre recombinase
Nuclearlocalizationsignal (NLS)
pAAV-CRE RecombinaseVector(6,115 bp)
hGHpolyA
Figure4. pAAV-CRERecombinasevectormap.
[Vector maps]
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Figure5. pAAV-LacZvectormap.
Figure6. pRC1vectormap.
Figure7. pRC2-mi342vectormap.
Ampr
pUCori ITR
ITR
CMVpromoter
LacZ
hGHpolyA
pAAV-LacZ Vector(7,622 bp)
Ampr
AAV2Rep
AAV1Cap
ColE1ori
pRC1 Vector(7,330 bp)
Ampr
ColE1ori AAV2
Rep
AAV2Cap
CMVpromoter
hsa-miR-342
HSV-TKpolyA
pRC2-mi342 Vector(8,189 bp)
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Figure10. pHelpervectormap.
Ampr
AAV2Rep
AAV5Cap
ColE1ori
pRC5 Vector(7,294 bp)
Ampr
AAV2Rep
AAV6Cap
ColE1ori
pRC6 Vector(7,330 bp)
Ampr
ColE1ori
AdenovirusVA
AdenovirusE4
AdenovirusE2A
pHelper Vector(11,635 bp)
Figure8. pRC5vectormap.
Figure9. pRC6vectormap.
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III. Storage-20℃StoreAAVExtractionSolutionAandAAVExtractionSolutionBatroomtemperatureafterthawing.Usewithin2yearsofreceipt.
IV. Materials Required but Not ProvidedIV-1. Equipment
• 100-mmdiametertissueculture-treateddishes• Generalequipmentforcellculture
IV-2. Reagents• TransfectionreagentCalPhos™MammalianTransfectionKit(Cat.#631312)Xfect™TransfectionReagent(Cat.#631317)
• Dulbecco'sModifiedEagle'sMedium(DMEM)4.5g/LGlucosewithL-Glutamine• FetalBovineSerum(FBS)• Trypsin-EDTA• AAVpro293TCellLine(Cat.#632273)*1• 0.5MEDTA(pH8.0)[EDTABufferPowder,pH8.0(Cat.#T9191)]• pAAV-ZsGreen1Vector(Cat.#6231)*2
*1 SeveralHEK293andHEK293Tcelllinesarecommerciallyavailable.Viralproductionishighlydependentonfeaturesofthecellline.AAVpro293TCellLine(Cat.#632273)andHEK293T/17cells(ATCC,CRL-11268)arerecommendedforpreparationofhigh-titerAAV.
*2 pAAV-ZsGreen1VectorisanAAVvectorplasmidthatexpressesthegreenfluorescentproteinZsGreen.UseasapositivecontroltoconfirmtheefficiencyoftransfectionandthetiterofthepreparedAAVparticles.
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V. Overview of AAV Particle PreparationPerformallstepsfromstepVI-1forAAVproHelperFreeSystem(AAV1/AAV2/AAV5/AAV6)(Cat.#6673,6230,6650,6651)tocloneaGOI.Fortheothersystems(Cat.#6668,6652,6653,6654,6669,6655,6656,6657),performstepsVI-3throughVI-7.
1. Cloningageneofinterest(GOI)intopAAV-CMVVector↓
2. Preparetherecombinantplasmid(pAAV-GOIvector)↓
3. CultureAAVpro293Tcells↓
4. Co-transfectAAVpro293TcellswithpAAV-GOI,pRC,andpHelpervectors↓
5. Changeculturemedium↓
6. CollectAAV-producingcells(2-3daysaftertransfection)↓
7. ExtractvirusparticlesfromAAV-producingcells
VI. ProtocolAAVproHelperFreeSystem(AAV1/AAV2/AAV5/AAV6)(Cat.#6673,6230,6650,6651),needstepVI-1andVI-2.Forotherkits(Cat.#6668,6652,6653,6654,6669,6655,6656,6657)skiptostepVI-3.
VI-1. Cloning a Gene of Interest into pAAV-CMV VectorInsertageneofinterest(GOI)intothemultiplecloningsite(MCS)ofthepAAV-CMVvectorusingstandardcloningmethods.TheIn-Fusion®HDCloningPluskit(Cat.#638909)canalsobeusedtoeasilyclonePCRproductsderivedfromtheGOIintoanylinearizedvector.Inaddition,theCMVpromotercanbereplacedwithanotherpromoterusingtheEcoRVsiteinthisplasmid(Figure3).
Note 1: TheGOIDNAfragmentshouldcontainanATGstartcodonandastopcodon.
Note 2: ThesizeoftheGOIinsertshouldbe<2.5kb.
VI-2. Preparation of the pAAV-GOI VectorAfterconfirmingthepresenceofthecorrectinsertinpAAV-GOI,prepareplasmidDNAusingaplasmidpurificationkit,suchasNucleoBondXtraMidi/Maxi(Cat.#740410.10/740414.10,etc.).AdjusttheplasmidDNAconcentrationto1μg/μl.Note: ThepurityofplasmidDNAisextremelyimportantforhightransfection
efficiency.
VI-3. AAVpro 293T Cell CultureInoculatea100-mmcellculturedishwith2.5-4.0x106AAVpro293TcellsinDMEMculturemediumsupplementedwith10%FBS.FortheCalPhosMammalianTransfectionKitortheXfectTransfectionReagent,10mlofthemediumshouldbeused.Cultureovernightaccordingtostandardcellcultureprotocols.
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VI-4. Transfection of AAVpro 293T Cells with pAAV, pRC, and pHelper Onedayafterplatingthecells,co-transfectwithapAAVvector(eitherpAAV-GOIvectorfromVI-2,pAAV-CRE,orpAAV-LacZ),pRCvector,andpHelpervector.
Fortransfection,theCalPhosMammalianTransfectionKit(Cat.#631312)orXfectTransfectionReagent(Cat.#631317)arerecommended;protocolexamplesforeachkitareprovidedbelow.
a. CalPhosMammalianTransfectionKit(Cat.#631312)ThefollowingprotocolismodifiedfromtheprotocolrecommendedforCalPhosMammalianTransfectionKit.FollowtheprotocolprovidedbelowtoobtainahightiterofAAVsolution.
1. Bring2XHEPES-BufferedSalinetoroomtemperature.2. Dilute2McalciumsolutionwithsterileH2O(includedinthekit)toobtaina333mMcalciumsolution(6-folddilution),andbringtoroomtemperature.
3. MixtheplasmidDNAandcalciumsolution.
pAAVVector 1μg/μl 6μlpRCVector 1μg/μl 6μlpHelperVector 1μg/μl 6μlCalciumSolution 333mM 1,000μlTotal 1,018μl
4. Addanequalvolumeof2XHEPES-BufferedSalineatroomtemperature.Closethelidofthetubeandvigorouslyshake15timestomix.
5. Allowtostandfor3min.Note: Adheretoastrict3-minincubationtime,thenproceedquicklyto
thenextstep.Withlongerincubation,largecalciumphosphate-DNAcomplexeswillformandtransfectionefficiencywilldecrease.
6. AddthemixturedropwisetotheculturedAAVpro293Tcells(fromStepVI-3)andculturethecellsfurther.Note: WiththeCalPhosMammalianTransfectionKit,itispossibletocheck
forcalciumphosphatecomplexesusingamicroscope.
b. XfectTransfectionReagent(Cat.#631317)1. VortextheXfectPolymer.2. MixtheXfectReactionBufferandtheplasmidDNA,andvortexvigorouslyfor5sec.
pAAVVector 1μg/μl 13μlpRCVector 1μg/μl 13μlpHelperVector 1μg/μl 13μlXfectReactionBuffer 561μlTotal 600μl
3. Add11.7μlofXfectPolymertotheplasmidmixture,andvortexvigorouslyfor10sec.
4. Allowtostandfor10minatroomtemperature.5. Centrifugethesolutionbriefly.AddthesolutiondropwisetotheculturedAAVpro293Tcells(StepVI-3)andculturethecellsfurther.
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VI-5. Change Culture MediumAtleast6hoursaftertransfection(upto25hours),completelyreplacetheculturemediumwithfreshDMEMcontaining2%FBS.
VI-6. Collection of AAV Particle-Producing Cells (2 - 3 Days after Transfection)1. Add1/80volumeof0.5MEDTA(pH8.0)toaculturemediumcontainingAAV-producingcellsandmixwell.Allowtostandatroomtemperaturefor10min.
2. Collectthedetachedcellsinasterile15-mlcentrifugetube.3. Centrifugeat1,750gat4℃for10min.Completelyremovethesupernatantandcollectthecellpellet.Note: Confirmthatthesupernatanthasbeencompletelyremovedbefore
proceeding;viralparticleisolationmaybeaffectedbyresidualsupernatant.
VI-7. Isolation of AAV Particles from AAV-Producing CellsTheuseoftheAAVExtractionSolutionincludedinthekitisstronglyrecommended.ThismethodyieldsAAVparticleswithhigherpurityandtiterthanstandardfreeze-and-thaworsonicationmethods(VIII.ReferenceData).
1. Loosenthecellpellet(fromstepVI-6)bytappingorvortexingthetube.Note: Ifthecellpellethasnotbeenloosenedsufficiently,theefficiencyofextraction
maydecrease.Confirmthattherearenoclumpsofcellsbeforeproceeding.2. Add0.5mlofAAVExtractionSolutionA.3. Suspendthecellpelletbyvortexingfor15sec.4. Allowtostandatroomtemperaturefor5min.Vortexfor15secagain.5. Centrifugeat2,000-14,000gat4℃for10mintoremovecelldebris.
Note: IfthetiteroftherecoveredAAVvectorislow,theefficiencymaybeincreasedbyrepeatingsteps3-5.
6. Collectthesupernatantinanewsterilecentrifugetubeandadd50μlofAAVExtractionSolutionBandmixbypipettingtoprepareAAVsolution.Note 1: TheAAVsolutioncanbestoredat-80℃.Thawquicklyina37℃water
bathbeforeuse.Note 2: ThesupernatantmaychangetoapinkcolorafterAAVExtraction
SolutionBisadded.
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VII. Measurement of Virus TiterVirustitercanbemeasuredbyreal-timePCR(vectorgenomeassay)orbyinfectionassay(biologicaltitermeasurement).Real-timePCRanalysisofvectorgenomesprovidesrapidquantification,whereasdeterminingtiterbyinfectionintocellsisgenerallymoreaccuratetodetermineinfectiousvirustiter.ThereareothertitrationmethodsforAAVvectorsthatinvolveassayofviralcapsidproteins,butthesemethodsmaydetectnonfunctional(empty)particles.
Vector Genome AssayTheAAVproTitrationKit(forRealTimePCR)Ver.2(Cat.#6233)canbeusedtomeasurevirustiterbyreal-timePCRanalysisusingtheviralITRdomainasatarget.
Biological Titer MeasurementThetiterisdeterminedbymeasuringtheexpressionofthegeneofinterest.TheprotocolbelowisatitrationmethodusingaAAV2vectorexpressingthefluores-centproteinZsGreen(pAAV-ZsGreen1Vector(Cat.#6231)).1. Preparetargetcellsatadensityof2-4x104cells/mlinDMEMwith10%FBS.2. Inoculateseveralwellsofa24-wellplatewith0.5mlofthecellsuspensionandcultureovernight.
3. PrepareserialdilutionsofthepreparedAAV2particlesolutionusingDMEMwith10%FBSandtheninfectthecellwiththedilutedvirussolution.Thedilutionratiodependsonthevirustiter,butserialdilutionsinthe1,000-100,000-foldrangearerecommended.
4. Threedaysafterinfection,detachthecellsusingTrypsin/EDTA,andanalyzeZsGreenexpressionbyflowcytometry.
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Figure11.EffectofmiRNA-342onAAV2production.
pRC2 pRC2-mi342
Totalvectorgenomes(vg)
4.5x1010
05.0x1091.0x10101.5x10102.0x10102.5x10103.0x10103.5x10104.0x1010
3.86x1010
1.89x1010
[Results]ThepRC2-mi342vectorresultedinatwo-foldincreaseintiter(vectorgenomes)ascomparedtopRC2.
VIII. Reference Data VIII-1. Increase in AAV2 Titer by the pRC2-mi342 VectorThepRC2-mi342vectorincludedincorrespondingkits(Cat.#6230,6652,6655)canbeusedtoproducehightiterrecombinantAAV2particles.
[Methods]VirusproducingCells:HEK293Transfection:CalciumphosphatemethodPlasmids:
•pAAV-CMV-AcGFP1Vector•pRC2-mi342VectororpRC2Vector*•pHelperVector
Culture:T25Flask*pRC2Vector: Vectorlackingthehsa-miR-342expressioncassette
AAV2particleswereextractedandthetiterwasevaluatedbyreal-timePCR.
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VIII-2. Efficiency of AAV Particle Extraction Using AAV Extraction SolutionTheAAVExtractionSolutionsAandBinthissystemcanbeusedtoeasilyandefficientlyextractAAVparticlesfromAAV-producingcells.
A. Comparison with Freeze-Thaw Method; Virus YieldHEK293cellsweretransfectedwiththepAAV-ZsGreen1vector(Cat.#6231)andcorrespondingplasmidsforeachAAVserotype.AAVparticlesexpressingZsGreen1wereextractedfromthecellsusingeitherAAVproExtractionSolutionorthefreeze-and-thawmethod.Thetiteroftheviralextractwasdeterminedusingthevectorgenomeassay(Figure12A)andbiologicaltitermeasurementwithHT1080cells(Figure12B).TheinfectiousAAVviruscanbeobtainedeasilyandefficientlyusingAAVproExtractionSolution.
Figure12A. AAVextractionefficiencyusingAAVproExtractionSolution(vectorgenomeassay).
AES:AAVproExtractionSolution F/T:Freeze-thawmethod *vg:vectorgenome
Genomictiter(vg* /ml)
Genomictiter(vg* /ml)
8.0x1010
01.0x10102.0x1010
4.0x10105.0x10106.0x10107.0x1010
9.0x1010
3.0x1010
AAV2
AAV50
1.0x10102.0x1010
4.0x10105.0x10106.0x10107.0x1010
3.0x1010
AAV60
2.0x1011
4.0x1011
8.0x1011
1.0x1012
1.2x1012
6.0x1011
4.8x1011
4.0x1011
3.2x1011
2.4x1011
1.6x1011
8.0x1010
AAV10
Figure12B. AAV2extractionefficiencyusingAAVproExtractionSolution(biologicaltiter).
ZsGreen1(%)
AES: AAVproExtractionSolutionF/T: Freeze-thawmethod
F/TAES
5
10
15
20
25
30
35
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B. Comparison with Freeze-Thaw Method; PurityAAVparticleswereobtainedfromHEK293producercellsusingAAVproExtractionSolutionorthefreeze-thawmethod.TheamountofviralgenomicDNAineachAAVextractwasquantifiedbyreal-timePCR.Then,theequivalentof1x109vgofeachAAVextractwasanalyzedbySDS-PAGEtoevaluatetheamountofproteinimpurity(Figure13).Inaddition,residualcellulardsDNAcontentineachAAV2extractwasassayedusingtheintercalationmethod(Figure14).TheresultsindicatethattheuseoftheAAVproExtractionSolutionclearlyreducedtheamountofproteinimpuritiesanddsDNAincomparisonwiththefreeze-thawmethod.
Figure13. SDS-PAGEofAAVextracts.ThepurityofAAVpreparedusingtheAAVExtractionsolution(AES)wasfargreaterthanthefreeze-thawmethod(F/T).
Figure14. dsDNAinAAV2extracts.ResidualdsDNAwaslesswhenAAV2waspreparedusingtheAAVproExtractionSolution(AES)ascomparedtothefreeze-thawmethod(F/T).
μg/1x1012 vg
Freeze-ThawMethod
AAVproExtractionSolution
1009080706050403020100
92.21
7.77
AES:AAVproExtractionSolution F/T:Freeze-and-ThawMethod (1x109vg/lane)
F/T AESAAV2
F/T AESAAV1
F/T AESAAV5
F/T AESAAV6
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VIII-3. Infection with AAV2-CRE particles AAV2-CREviralparticleswerepreparedusingtheAAVproHelperFreeSystem(AAV2-CRERecombinase)(Cat.#6652)andpurifiedusingtheAAVproPurificationKit(AAV2)(Cat.#6232).ParticleswereusedtoinfectHEK293cellsthatareengineeredtofluoresce(ZsGreen1)whenrecombinationoccurswithCrerecombinase.TheproportionoffluorescentcellscorrelatedpositivelywiththeamountofAAV2-Creparticlesusedforinfection.
Figure15.FACSanalysisofHEK293cellsinfectedwithAAV2-CREparticles.
Uninfected(0.07%)
100vg/cell(30.81%)
12,500vg/cell(92.05%)
100
80
60
40
20
0101 102 103 104 105
ZsGreen1
%ofMax
VIII-4. Infection with AAV2-LacZ ParticlesAAV2-LacZviralparticleswerepreparedusingtheAAVproHelperFreeSystem(AAV2-LacZ)(Cat.#6655)andpurifiedusingtheAAVproPurificationKit(AAV2)(Cat.#6232).ParticleswereusedtoinfectHT1080cells.StainingwasperformedusingtheBeta-GalactosidaseStainingKit(Cat.#631780).
Figure16.X-galstainingofHT1080cellsinfectedwithAAV2-LacZparticles.
AAV2-LacZNC
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IX. References1) Miyake,etal .JNipponMedSch.(2012)79(6):394-402.2) VanVliet,etal. MethodsMolBiol. (2008)437:51-91.3) Wu,etal .MolTher. (2006)14(3):316-27.4) Zincarelli,etal .MolTher. (2008)16(6):1073-80.5) Ellis,etal.VirolJ .(2013)10:74.
X. Related ProductspAAV-ZsGreen1Vector(Cat.#6231)AAVpro®PurificationKit(AllSerotypes)(Cat.#6666)AAVpro®PurificationKit(AAV2)(Cat.#6232)AAVpro®TitrationKit(forRealTimePCR)Ver.2(Cat.#6233)AAVpro®ExtractionSolution(Cat.#6235)AAVpro®PackagingPlasmid(AAV1)(Cat.#6672)AAVpro®PackagingPlasmid(AAV2)(Cat.#6234)AAVpro®PackagingPlasmid(AAV5)(Cat.#6664)AAVpro®PackagingPlasmid(AAV6)(Cat.#6665)CalPhos™MammalianTransfectionKit(Cat.#631312)Xfect™TransfectionReagent(Cat.#631317/631318)AAVpro®293TCellLine(Cat.#632273)Beta-GalactosidaseStainingKit(Cat.#631780)
AAVproisaregisteredtrademarkofTAKARABIOINC.In-FusionisaregisteredtrademarkofTakaraBioUSA,Inc.CalPhosandXfectaretrademarksofTakaraBioUSA,Inc.
NOTE : Thisproductisforresearchuseonly.Itisnotintendedforuseintherapeuticordiagnosticproceduresforhumansoranimals.Also,donotusethisproductasfood,cosmetic,orhouseholditem,etc.Takaraproductsmaynotberesoldortransferred,modifiedforresaleortransfer,orusedtomanufacturecommercialproductswithoutwrittenapprovalfromTAKARABIOINC.Ifyourequirelicensesforotheruse,pleasecontactusbyphoneat+81775656973orfromourwebsiteatwww.takara-bio.com.Youruseofthisproductisalsosubjecttocompliancewithanyapplicablelicensingrequirementsdescribedontheproductwebpage.Itisyourresponsibilitytoreview,understandandadheretoanyrestrictionsimposedbysuchstatements.Alltrademarksarethepropertyoftheirrespectiveowners.Certaintrademarksmaynotberegisteredinalljurisdictions.
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