Download ORF manual_20160301 - Vigene Biosciences

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
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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.
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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
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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:
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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:
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pDEST-PGK
AlthoughCMVpromoterusuallyisverystrongindrivinggeneexpressioninvitroand
invivo,therearesomereportssuggestedCMVpromotercouldbesilencedfrom
unknownreason.Inordertoaddressthisproblem,ViGeneprovidesdestination
vectorsPGKorEF-1apromoter.Bothpromotershavebeenreportedtohavegood
expressionforinvivoandsomecelllineswhereCMVissilenced.InpDest-PGKvector,
amplicillinselectionmarkerreplacedthekenamycinselectionmarkerandPGK
promoterreplacedCMVpromoterinpEnter.OtherelementsandMCSremainthe
sameinbothvectors.
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pDEST-C-GFP
Fluorescentproteintagsareveryusefulintracingproteinexpressionandlocationin
vitroandinvivo.InadditiontodestinationvectorswithFlag,His,HA,Myctags,
ViGeneprovidesdestinationvectorswithfluorescentproteintags,suchasGFPand
RFP.AlldestinationvectorsfromViGeneBiosciencesarewithampicillinselection
markerandwithdifferenttagseitherasN’terminalfusionorC’terminalfusion.
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LentiviralDestinationvectors
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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
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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
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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.
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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.
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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
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Volume
5µl
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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.
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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.
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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
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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
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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
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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.
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LIMITEDPRODUCTWARRANTY
Thiswarrantylimitsourliabilitytoreplacementofthisproduct.Nootherwarranties
ofanykind,expressorimplied,includingwithoutlimitation,impliedwarrantiesof
merchantabilityorfitnessforaparticularpurpose,areprovidedbyViGene
Biosciences.ViGeneBiosciencesshallhavenoliabilityforanydirect,indirect,
consequential,orincidentaldamagesarisingoutoftheuse,theresultsofuse,orthe
inabilitytousethisproduct.
ORDERINGINFORMATIONANDTECHNICALSUPPORT
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TollFree(USA):1-800-485-5808
Telephone:301-251-6638
Fax:301-251-6110
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