7QSP )FMQFS'SFF 4ZTUFN - Takara Bio · Viruses produced with AAV-based vectors could, depending on...

Cat. # 6230, 6650 - 6657,6668, 6669, 6673

Product Manual

AAVpro® Helper Free System

For Research Use

v201611Da

【 For China/Korea/India/Europe 】

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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v201611Da

URL:http://www.takara-bio.com

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).


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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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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




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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


Ampr

AAV2Rep

AAV6Cap

ColE1ori


Ampr

ColE1ori

AdenovirusVA

AdenovirusE4

AdenovirusE2A

pHelper Vector(11,635 bp)



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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

0


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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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