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FullLengthcDNAClones& ORF-AdenoviralExpressionSystem UserManual Revision201603.01 ©ViGeneBiosciences2016 RESEARCHUSEONLY. Notforuseindiagnosticprocedures Thisproductshallbeusedbythepurchaserforinternalresearchpurposeonlyand redistributionisstrictlyprohibitedwithoutwrittenpermissionfromViGene BiosciencesInc. TableofContent Components ......................................................................................................... 3 Storage ............................................................................................................... 3 SafetyConsiderations ............................................................................................. 3 Introduction ......................................................................................................... 4 pEnter vector map and features: ....................................................................... 5 Most common destination vectors and features: ................................................. 6 pDEST-PGK .................................................................................................. 7 pDEST-C-GFP ............................................................................................... 8 Lentiviral Destination vectors ......................................................................... 9 Destination of Adeno-Associate Vrial (AAV) vector ......................................... 11 pAD vectors ................................................................................................ 11 Transfer ORF insert from pEnter to destination vector: ..................................... 12 Recombination: .......................................................................................... 14 Adenovirus packaging: ................................................................................ 14 pEnter-ORF plasmid amplification:................................................................ 15 Viral vector amplification: ............................................................................ 15 Titering Adenovirus with quantitative-PCR: ................................................... 16 FAQ: ................................................................................................................. 17 LIMITEDPRODUCTWARRANTY .............................................................................. 21 ORDERINGINFORMATIONANDTECHNICALSUPPORT ................................................. 21 Ordering......................................................................................................... 21 TechnicalSupport ............................................................................................ 21 ©ViGene Biosciences www.vigenebio.com Page 2 Components Theproductsmaycontainthefollowingcomponents. 1. Catalognumber(CHXXXXXX):pEnter-ORFglycerolstock.500ul. 2. Catalognumber(AHXXXXXX):pAD-ORFglycerolstock.500ul. 3. Catalognumber(VHXXXXXX):ORF-adenovirus:one100ulvialof10^9adenoviralparticles. Storage Theproductsshouldbestoredat-80ºC. SafetyConsiderations FortheORFadenoviruscustomers-followtherecommendedNIHguidelinesforall materialscontainingBSL-2organisms. FortheORFcDNAclonecustomers-followtherecommendedNIHguidelinesforall materialscontainingBSL-1reagents. ©ViGene Biosciences www.vigenebio.com Page 3 Introduction ViGene'sfulllengthcDNAclonesfeatureproprietarycloningtechnologyand uniquevectordesign-pEnterEntryVectorSystem.Theexpression-readyORFscan beshuttledintomorethan30destinationvectorsinasimplecut-and-paste approachinjust2-3hours. Keyfeatures •100%sequence-verifiedbyNextGenSequencing •Expressionguaranteed •Readytobeshuttledintomorethan30destinationvectors,including adenoviral&lentiviralvectors •Containspuromycingeneforstablecelltransfection •UniquedesigntoaccommodatecDNAupto30kb •TheCMVpromoterandaKozakconsensussequencedrive proteinexpressioneffectively ViGeneBiosciences’ORF-AdenoviralORFexpressionsystemcontainsthreesetsof relatedproducts:ORFshuttlingsystem,pAD-ORFandpremadeORF-Adenovirus. Recombinantadenovirusesarepowerfulandeasy-to-usetoolsingenedeliveryand expression.Severaluniquecharactersofadenoviralbiologyhavemadeitthevector ofchoiceforbroadapplication.First,itiscapableofinfectingvarietiesofcelltypes, includingdividing,non-dividingcellsandstemcells.Moreover,highvirustitercanbe easilyobtained.Furthermore,hightiterofviruscouldachievehighinfectionrateand highexpressionlevel.Lastbutnottheleast,afterenteringthecells,thevirus remainsepichromosomal,thustheexpressionistransientandinfectionof recombinantadenovirusdoesnotinducechromatinchangeinhostcell.InViGene Biosciences,weusedthemostcommonadenoviralvector,humanadenovirus serotype5,whichisrenderedreplicationdefectivebythedeletionofE1andE3 genes.TheE1geneisessentialfortheassemblyofinfectiousvirusparticles,andit canbecomplementedduringviruspackagingprocessinHEK293Tcelllines.Andthe E3geneisdispensable.WiththedeletionofE1andE3,adenoviralparticlesare capableofintegrateof7.5kbforeignDNA. ORFplasmidshuttlingsystem.Utilizingsimple“cutandpaste”cloningstrategy, ViGeneprovidesvarietiesofchoicestoexpressORFwithoneortwodifferent epitopetagsorfluorescentmarkers.Withfourdifferentcombinationsof endonucleasewithraresitesinsidegenes,andwithdifferentselectionsbetween entryvectoranddestinationvectors,ORFinsertscouldbeeasilytransferredfrom ©ViGene Biosciences www.vigenebio.com Page 4 theentryvectortoanyofthedestinationvectorsintwodays.Therearemorethan 30destinationvectorstohelpcustomerstoachievethebestmethodsinoverexpress theORFs.ForthedetailedlistofViGene’sdestinationvectors,pleasevisitourweb site,www.ViGeneBio.com.Thesedestinationvectorsincludesvectorswithdifferent promoters,viralvectorsofadenovirus,lentivirusandadeno-associatedvirus,vectors totagtheepitopeorfluorescentmarkerattheN’terminalorC’terminalofORFs.In ViGene’spEntervector,expressionofORFisdrivenbyCMVpromoter,iswithFlag andHistagattheC’terminusofORF,andwithaSV40poly(A)tailingsignal.pENter containsapuromycinmarkerdrivenbySV40promoter,whichcanbeusedto generatestabletransfection.pEnteralsocontainstwoadenoviralITRsequenceand twoAd5homologoussequences,whichcanbeusedtotransferORFexpressionunit toadenoviralvector,pAD. pAD-ORFisplasmidbasedORFofgeneexpressionsystem.Throughrecombination inE.coli,theORFexpressionunitwastransferredfromthepEnterandmostofthe destinationvectorstopAD,exceptthedestinationvectorsoflentiviralandadenoassociateviralvectors.pADismostcommonlyusedhumanadenoviralvector, humanadenovirusserotype5,withE1andE3genedeletion.PlasmidofpAD-ORFs couldbeusedtogeneraterecombinantadenovirusinHEK293cells. ORF-Adenovirusispremaderecombinantadenovirus.Theadenoviralvectoris generatedthroughtherecombinationofORFcarryingpEnterandpAD.Theexpress ORFsarewithFlagandHistagsattheirC’termini.ViGene’spremadeORFAdenovirusprovidesthemostefficientmodeofgenedeliveryamongallformsof ORFcDNAclones.ItcanbeusedinvitroandinvivoforORFfunctionalanalysis. pEnter vector map and features: ©ViGene Biosciences www.vigenebio.com Page 5 Inmostofcase,ORFinsertsareclonedbetweenAsisIandMluIsites.Inotherrare casethecombinationofAsisI-RsrII,AsisI-NotIorAscI-MluIareusedinthecloning. PleasecheckourwebsiteortheCOAforspecificclones.InthepEntervector,ORFis fusedwithaFlag/Histagatitscarboxylterminus.Thevectorcontainsakanamycin markerforbacterialselection.TheSV40drivenpuromycinmarkercanbeusedfor stablecloneselectioninmammaliancells.TwoITRsandtwoArmsequencesare designedtogeneraterecombinantadenoviralvectorbyrecombinationwithpADin E.coliandadenovirusinHEK293cells.ViGene’spEntervectorisamammalianORF expressionvector,dualtagsofFlagandHiscouldbeusedtodetectandpurify proteinsexpressedinmammaliancells. Most common destination vectors and features: ©ViGene Biosciences www.vigenebio.com Page 6 pDEST-PGK AlthoughCMVpromoterusuallyisverystrongindrivinggeneexpressioninvitroand invivo,therearesomereportssuggestedCMVpromotercouldbesilencedfrom unknownreason.Inordertoaddressthisproblem,ViGeneprovidesdestination vectorsPGKorEF-1apromoter.Bothpromotershavebeenreportedtohavegood expressionforinvivoandsomecelllineswhereCMVissilenced.InpDest-PGKvector, amplicillinselectionmarkerreplacedthekenamycinselectionmarkerandPGK promoterreplacedCMVpromoterinpEnter.OtherelementsandMCSremainthe sameinbothvectors. ©ViGene Biosciences www.vigenebio.com Page 7 pDEST-C-GFP Fluorescentproteintagsareveryusefulintracingproteinexpressionandlocationin vitroandinvivo.InadditiontodestinationvectorswithFlag,His,HA,Myctags, ViGeneprovidesdestinationvectorswithfluorescentproteintags,suchasGFPand RFP.AlldestinationvectorsfromViGeneBiosciencesarewithampicillinselection markerandwithdifferenttagseitherasN’terminalfusionorC’terminalfusion. ©ViGene Biosciences www.vigenebio.com Page 8 LentiviralDestinationvectors ©ViGene Biosciences www.vigenebio.com Page 9 Lentiviralvectorsarevaluabletoolsindeliveringgeneinvitroandinvivo.Compared toadenovirus,lentivirushastheabilitytointegrateforeigngenesintothegenomeof non-dividingcells.ViGeneBiosciencesprovideslentiviralvectorswithveritiesoftags andfluorescentmarkers.AspLent-Flag-His,mostofourlentiviralvectorshavethe similarMCSasourpEntervectorandwithampicillinselectionmarker.AnyORFgene canbeeasilytransferredfrompEntervectortoourlentiviralvectorsby“cutand paste”cloningmethod. Lentiviralvectorscanbeeasilyusedtogeneratestabletransfectioninvitrowhen comparedtoplasmidDNA.Tofacilitatetheselectionofstabletransfection,we providetwolentiviralvectorswitheitherpuromycinorGFPselectionmarker,which isdirectlydrivenby5’LTRpromoterforexpression.Inthistwovectors,Customers canchoosetoexpresstheORFgenewithamyc-flagtagbytransferringtheORF insertsfrompEnterby“cutandpaste”method.IfCustomerschoosetoexpressthe ©ViGene Biosciences www.vigenebio.com Page 10 ORFgenewithnativestopcodon,theORFinsertsfromourpEntercanbePCRedout andcloneintothedestinationvector,asa“copyandpaste”cloningmethod. DestinationofAdeno-AssociateVrial(AAV)vector WhencomparedtoAdenovirus,AAVisasmallvirusanditcausesverymildimmune response.MostlabschoseAAVasgenetherapyvectorsorinvivoanimalresearch. AAVcandelivergeneintobothdividingandquiescentcells.Sameasadenovirus, afterenteringthecells,theAAVvirusremainsepichromosomal.Toaccommodate theneed,ViGeneBiosciencesprovidesoneAAVvectorforORFgeneexpression. pAV-FHdrivestheORFgenesexpressionbyCMVpromoter,withSV40polyAtail.The expressedORFsarewithaC’terminalfusedFlag-Histag.ORFinsertscanbe transferredfrompEntertopAV-FHbyour“cutandpaste”cloningmethod. pADvectors ©ViGene Biosciences www.vigenebio.com Page 11 34200bp 34200bp pADvectorscontainshumanAd5adenoviralgenomesequencewithE1andE3 deletion.InordertotransfertheORFgenesintopADvectors,researcheshavecarry outrecombinationbetweenpADvectorsandthelinerDNAofeitherORFinpEnter orinotherdestinationvectorsinrecombinationcompetentE.coli,suchasBJ5183 cells.InViGeneBiosciences,wegeneratedtwopADvectors.ThepAD-Amphasthe ampicillinselectionmarker,andisusedinrecombinationwithpEntervector.The pAD-Kanhaskanamycinselectionmarker,andisusedinrecombinationwith destinationvectors. Transfer ORF insert from pEnter to destination vector: WithinViGeneBiosciencesORFplasmidshuttlingsystem,ORFinsertscouldbeeasily transferredfromtheentryvectortoanyofthedestinationvectorsintwodays.The transferprocedureisillustratedinfollowingdiagram. ©ViGene Biosciences www.vigenebio.com Page 12 1.Thedestinationcouldbepreparedinadvance,orpurchasethereadytouse destinationvectorsfromViGeneBiosciences. 2.DigesttheORFinsertsfrompEnterwithrightenzymecombination,forexample,the mostcommonAsisI-MluIcombinationinMixtureI: Component Volume RestrictionDigestBuffer 3µl ORFinpEnter 5µl(500ng) AsisI 0.6µl MluI 0.6µl Nuclease-FreeWater 20.8µl 3.Thedigestioncanbedonein5minutesat37ºC.Thentheenzymesshouldbe inactivatedin80ºCfor15minutes. 4.SetupligationreactionasinMixtureII: Component Volume 5xRapidLigationBuffer 2µl MixtrueI 2µl(30ng) Pre-preparedOriGeneVector 3µl(10ng) T4DNALigase(400/µl) 0.5µl Nuclease-FreeWater 2.5µl 5.Ligationreactionisdoneatroomtemperaturefor5minutes.Andalltheenzymescan bepurchasedfromNewEnglandBiolabs. 6.TransformandincubateE.colicellsat37ºCovernight. 7.Day3,DNAminiprepation,DNAdigestionorsequencingtoscreenforcorrect clones. ©ViGene Biosciences www.vigenebio.com Page 13 Recombination: UsingeitherthepEnterordestinationvectorsasshuttlevectors,pAD-ORFclonescan begeneratedinrecombinationcompetentE.coli,suchasBJ5183cells. 1.DigestthepEnter-ORFwithPmeIenzyme. Component Volume RestrictionDigestBuffer 5µl ORFinpEnter 7µl(700ng) PmeI(fromNEB) 1µl Nuclease-FreeWater 37µl 2.Incubateat30ºCfor2.5hourthenadd0.5µlAlkalinephosphatase,CalfIntestinal (CIP)andincubateat37ºCfor30minutes. 3.Thedigestionwillgeneratetwofragments,oneat1.2kbandtheotheroneshould belargerthan6.3kbdependentonthesizeofORFinserts.Separatethetwo fragmentsbyDNAagarosegelelectrophoresis. 4.RecoverthelargerfragmentwithDNAgelextractionkit.Note:elutetheDNAwith 30µlwaterinsteadofTEbufferatthelaststep. 5.Transform30µlofBJ5183electricalcompetentcellswith5µlofrecoveredDNAby electroporation. 6.Afterelectroporation,resuspendthecellsin800µlofSOCmediumandletthecell recoverfor2hoursat32ºCshakingat250-290rpm. 7.Plate150µloftransformationonLB-Agarplatewith30µg/mlkanamycinand cultureat30-32ºCfor24hours. 8.Pool20-30coloniesfromtheplatein1-2mlLBmediumwith30µg/mlkanamycin andculturefor2-3hoursat32ºC. 9.IsolateplasmidDNAfromtheculturethentransform1µlofpreparedDNAtoDH5a andcultureonLB-Agarplatewith30µg/mlkanamycinat37ºCforovernighttogetrid ofpAD-Ampplasmid. 10.Pickatleast6coloniestoscreenforrightrecombination. 11.VerifytherightrecombinationbyPacIdigestionandDNAsequencing.Dependent thesitesofrecombination,correctrecombinationwillgeneratetwofragmentseither 3.5kband34kbor2.5kband34kb. Adenoviruspackaging: 1.DigestthepAD-ORFwithPacIenzyme. Component RestrictionDigestBuffer ©ViGene Biosciences Volume 5µl www.vigenebio.com Page 14 ORFinpEnter 20µl(2µg) PacI(fromNEB) 1µl Nuclease-FreeWater 24µl 2.Incubateat37ºCfor3hour.Thentheenzymesshouldbeinactivatedin80ºCfor 15minutes. Note:ThefollowingprocedureissuggestedforT75flasksandmaybeoptimizedtosuit individualneeds. 3.Seed3-5x106HEK293TcellsinaT75flaskonedaybeforetransfection. 4.Transfectcellswith2uglinerpAD-ORFDNA. 5.4-5daysaftertransfection,examinethemonolayertwiceperdayunderthe microscopeforCPE.WhenCPEisnearlycomplete(i.e.mostcellsroundedbutnotyet detachedfromtheflask,usuallyittakes7-14daysforCPEtobecomplete),harvestcells bypipettingmediaupanddowntowashtheinfectedcellsfromtheflaskintothemedia. 6.Poolcellsandmedium.Pelletcellsbycentrifugationat1000gfor5minutes.Remove supernatant,resuspendcellpelletinmediumorin10mMTris,pH8.0,100mMNaCl. (0.25-0.5mLperT75flask). 7.Releasetheadenovirusesfromthecellsuspensionwiththreefreeze/thawcycles. Centrifugeat3000gfor10minutestopelletthecelldebris.Discardthepelletandsave supernatantasviralstock. 8.Theviralsupernatantcanbestoredat-80°Corimmediatelypurifiedortitered. pEnter-ORFplasmidamplification: GlycerolstockofpEnter-ORForpAD-ORFshouldbestreakedonLB-Agarplatewith 30ug/mlkanamycinandcultureat37ºCovernight.Secondday,atleast3colonies shouldbepickedforminipreparation.ThesequencingpEnter-ORForpAD-ORFfrom the5’endshouldbeperformedwithaprimerwhoseprimingsiteislocated~150nt upstreamofthepolylinker. Viralvectoramplification: AmplificationofavirusstockisachievedbyinfectionHEK293Tcultureswith includedMirAdmicroRNAprecursoradenovirus.Oneroundofamplification generallyproducesa10-foldincreaseintiter. Note:ThefollowingprocedureissuggestedforT75flasksandmaybeoptimizedtosuit individualneeds. ©ViGene Biosciences www.vigenebio.com Page 15 1.Seed3-5x106cellsinaT75flaskonedaybeforeinfection. 2.Add50%oftheaboveCrudeViralLysatetothecultuire.Werecommendusinga multiplicityof>0.5PFU(plaqueformingunits)orenoughvirusesthatcellsdemonstrate cytopathiceffects(CPEs)within48hrs. 3.During24-48hrinfection,examinethemonolayertwiceperdayunderthe microscopeforCPE.WhenCPEisnearlycomplete(i.e.mostcellsroundedbutnotyet detachedfromtheflask),harvestcellsbypipettingmediaupanddowntowashthe infectedcellsfromtheflaskintothemedia. 4.Poolinfectedcellsandmedium.Pelletcellsbycentrifugationat1000gfor5minutes. Removesupernatant,resuspendcellpelletinmediumorin10mMTris,pH8.0,100mM NaCl.(0.25-0.5mLperT75flask). 5.Releasetheadenovirusesfromthecellsuspensionwiththreefreeze/thawcycles. Centrifugeat3000gfor10minutestopelletthecelldebris.Discardthepelletandsave supernatantasviralstock. 6.Theviralsupernatantcanbestoredat-80°Corimmediatelypurifiedortitered. TiteringAdenoviruswithquantitative-PCR: qPCRmethodisasimpleandhighthroughputmethodinestimatingadenoviral particlesinbothcrudelysateandpurifiedadenovirussamples.Thismethodisbased onthequantitativereal-timePCRamplificationofspecificadenoviralgenome sequence.Withinthelinearrangeofquantification,theinitialcopynumberofviral genomecanbeestimatedwhentheCtiscomparedwiththeCtfromknowncopy numberplasmid. 1.AdenovialgenomicDNApurification: Adenoviralvirions’DNAgenomeissurroundedbyacapsidofstructuralproteins.Itis preferredtodisruptthisproteinshellwithproteinaseK. Component Volume Viralsample 5µl ProteinaseK(5µg/µl) 1µl Nuclease-FreeWater 4µl I. Incubateat37ºCfor30minutes.Thentheenzymesshouldbe inactivatedin95ºCfor20minutes. II. Centrifugeat3000gfor10minutes,savethesupernatant. 2.QPCR: 1.TemplatestandardfromViGeneBiosciencesis3.6X10^8copies/ml.Seriesdilutethe standardto3.6×102-3.6×108andusenuclease-freewaterasnegativecontrol. ©ViGene Biosciences www.vigenebio.com Page 16 2.SetupPCRreaction,20µlforeachsample. Component Volume 2XSaberGreenMix 10µl Primermix 1µl Nuclease-FreeWater 7µl Treatedviralsampleorstandard 2µl(equivalentto1µlinitialviralsample) Samplesettingshouldbeasinfollowingtable. STD STD STD STD STD 2 3 4 0 3.6×10 3.6×10 3.6×10 3.6×105 STD STD STD STD STD 2 3 4 0 3.6×10 3.6×10 3.6×10 3.6×105 Viral Viral Viral Viral Viral sample1 sample1 sample2 sample2 sample3 STD 6 STD STD 3.6×10 3.6×10 3.6×108 STD STD STD 6 3.6×10 7 7 3.6×10 3.6×108 Viral Viral Viral sample3 sample4 sample4 3.PCRprotocol 4.Viralparticlesestimation. ViralParticles(particles/ml)=EstimatednumberfromstandardX1000 FAQ: 1. DoeshumanORFIpurchasedfromViGeneBiosciencesexactly matchthereferencesequence? No,itdoesnotalwaysmatchtoRefseq.AlthoughmosthumanORFsprovidedbyViGene BioSciencesmatchthereferencesequencepostedbyNCBI,someORFsfromViGene BioSciencescontainsinglenucleotidespolymorphisms(SNP),smallinframeinsertionsor deletions.HumanORFsfromViGeneBiosciencesweresubclonedeitherfromclonesin MammalianGeneCollection(MGC),orfromcDNAlibrary,thedifferencemayreflectthe differencefromdifferenttissueanddifferentindividuals. 2. AreallhumanORFsfromViGeneBiosciencesfullysequenced? Yes,allthehumanORFclonesfromViGeneBiosciencesarefullysequenced.Eachclone wasfirstconfirmedbyendssequencesfromSangersequencing,thenwassequencedby nextgenesequencing.Theactualclonesequenceispostedonourwebsite, www.vigenebio.com.AnysequencedifferencebetweenViGene’scloneandthe referencesequenceisalsoposted.Beforeorder,westronglyencouragecustomersto downloadoursequenceandcheckthedifferences. 3. Whatisthesafetyconsiderationwhenusingandhandling ©ViGene Biosciences www.vigenebio.com Page 17 adenoviralvector? mirAdadenoviralvectorisreplicatingdeficienthumanadenovirusserotype5and withdeletionoftheE1andE3genes.Thebiosafetyofficeatyourinstitution mustbenotifiedpriortouseofadenoviralvectorforpermissionandforfurther institution-specificinstructions.BL2conditionsshouldbeusedatalltimewhen handlingtheviralparticles.Alldisinfectionstepsshouldbeperformedusingfresh 10%bleach.Glovesshouldbewornatalltimeswhenhandlingadenoviral particlespreparationsandtransducingcells. 4. HowdoIdeterminewhetheradenoviralvectorcouldbeusedfor mygenedelivery? Youshouldtakefollowingconsiderationbeforeyouchoseadenoviralvector.1. Doyouneedtransientorstablegeneexpression?2.Doyouneedtotransduct dividingornon-dividingcells?3.Howimportantispotentialimmuneresponse frommytargetcell?4.Willyouuseviralparticlesforinvivoorinvitrogene delivery? Adenoviralvectorcaninfectdividingandnon-dividingcells.Itissuitablefor transientgeneexpressionwithhighgenedeliverefficiency,goodforinvivoand invitrogenedelivery.ButAdenoviralvectorhasrelativehigherimmune responseintargetcellswhencomparedtootherviralvectorsystem.Pleaserefer tothistableforchoosingyourviralvectorsystem. Adenovirus Adeno-associated Lentivirus Virus Genome dsDNA ssDNA ssRNA(+) Coat Naked Naked Enveloped Genomesize 38-39kb 5kb 9kb Infection/tropism Dividingandnon- Dividingandnon- Dividingandnondividingcells dividingcells dividingcells HostGenome Non-integrating Non-integrating Integrating Interaction Transgene Transient Potentiallong Longlasting expression lasting PackagingCapacity 7.5kb 4.5kb 6kb ImmuneResponse High VeryLow Low RelativeViralTiter 10E11without 10E12without 10E7without purification concentration concentration Relative 100% 70% 70% Transduction Efficiency RelativeForeign High Meidum Meidum GeneExpression ©ViGene Biosciences www.vigenebio.com Page 18 5. WhatisthedifferencebetweenVP,PFU,andIFU?Whichone shouldIuseindesigningexperiment? Viralparticles(VPs)representthetotalnumberofviralparticles(includinglive anddeadones).Thepercentageofdeadviralparticlescanvariessignificantly becauseofthevariationsinviruspreparation.ThusVPcannotbeusedin estimatingfunctionalvirusamount. PFU(plaqueformationunit)reflectstheamountofinfectiousviruses.Inmost cases,theVP/PFUratiois20:1to50:1.IFU(infectiousunit)isequivalenttoPFU. WesuggestthatyoushouldusePFUandIFUinyourexperimentaldesigninorder togetmoreconsistentresults. 6. .Whatarethemethodsindeterminetheviraltiter? Therearethreemostlyusedmethodsindeterminetheadenoviraltiter.1.Plaque formationassay;2.End-pointdilutionassay;and3.BCAassay. Plaqueformationassaymeasuretheconcentrationofinfectiousviruses(PFU), andisabiologicalassay.Plaqueformationassayisdoneonamonolayerof HEK293cellsthatareinfectedwithdilutedviruses.Viruseswillpropagatein infectedcells,causecytotoxicityeffectsandgetreleasedfrominfectedcells.The releasedviruswillinfectedneighboringcells,andthewholeprocesswillbe repeatedandleadtoformholesorplaquesonthecellmonolayer.0.5%agarose islaidontopofcellsaftertheinitialinfectiontopreventthediffusionofthe viruses.Viraltiter(PFU/ml)canbecalculatedbasedontheplaquesobserved. Plaqueformationassaytakeupto3weeks. End-pointdilutionassayisverysimilartoplaqueformationassay,butmuch complicate.Theformulaforcalculatingthevirustiterisalsocomplicate. BCAassaysmeasuretheconcentrationofProtein.Itisonlygoodforpurified adenoviralvectors.Ingeneral1ugprotein=1X109VP. 7. ShouldIpurifyandconcentratetheviralparticleswithCsClgradient ultracentrifugation? Iftheviralparticlesareusedininvitrocellcultures,doubleCsClpurificationis notrequired.However,thecontaminantsfromcelldebrisandculturemedium ©ViGene Biosciences www.vigenebio.com Page 19 contributegreatlytotheimmuneresponses.Viralparticlesusedininvivoor animalstudiesshouldbepurifiedandconcentratedbyCsClpurificationinorder toremovedefectiveparticlesandothercontaminants. 8. Howtostoreadenoviralparticles? Theviralparticlesarestableat-80°Cfor6monthstoayear. 9. WhatistheserotypeofmirAdadenoviralparticles? MirAdadenoviralparticlesarehumanadenovirusserotype5.WithE1/E3gene deletion. 10.WhatisRCAsandhowtodetectRCAs? The293celllinecontainsadenovirustype5sequenceswhicharehomologous withsequencesinmirAdadenoviralvector;Therefore,itispossiblethat replicationcompetentadenoviruses(RCA)canemergeasaresultoftherare recombinantofadenovirusandthegenomeofHEK293cells. TheRCAcanbetestedinnonpermissivecelllinessuchasHeLa.Serialdiluted adenoviralstocksareusedtoinfectHeLacellsandtheinfectedcellsare incubatedfor8days.Ifafter8daysnocytopathologyisapparent,recombinant adenoviralstockscanbeassumedtobefreeofRCA. ©ViGene Biosciences www.vigenebio.com Page 20 LIMITEDPRODUCTWARRANTY Thiswarrantylimitsourliabilitytoreplacementofthisproduct.Nootherwarranties ofanykind,expressorimplied,includingwithoutlimitation,impliedwarrantiesof merchantabilityorfitnessforaparticularpurpose,areprovidedbyViGene Biosciences.ViGeneBiosciencesshallhavenoliabilityforanydirect,indirect, consequential,orincidentaldamagesarisingoutoftheuse,theresultsofuse,orthe inabilitytousethisproduct. ORDERINGINFORMATIONANDTECHNICALSUPPORT Ordering • • • • Email:[email protected] TollFree(USA):1-800-485-5808 Telephone:301-251-6638 Fax:301-251-6110 TechnicalSupport • • • • Email:[email protected] TollFree(USA):1-800-485-5808 Telephone:301-251-6638 Fax:301-251-6110 ©ViGene Biosciences www.vigenebio.com Page 21