The use of viral 2A sequence for the simultaneous over expression of both the vgf gene and enhanced green fluorescent protein eGFP

ContentslistsavailableatScienceDirect

Journal

of

Neuroscience

Methods

j ou rn a l h o m epa g e :w w w . e l s e v i e r . c o m / l o c a t e / j n e u m e t h

Basic

neuroscience

The

use

of

a

viral

2A

sequence

for

the

simultaneous

over-expression

of

both

the

vgf

gene

and

enhanced

green

fluorescent

protein

(eGFP)

in

vitro

and

in

vivo

Jo

E.

Lewis

_,

_John

_M.

_Brameld

_,

_Phil

_Hill

_,

_Perry

_Barrett

_,

_Francis

_J.P.

_Ebling

_,

Preeti

H.

Jethwa

a_{DivisionofNutritionalSciences,SchoolofBiosciences,UniversityofNottingham,SuttonBoningtonCampus,LoughboroughLE125RD,UK} b_{SchoolofLifeSciences,UniversityofNottinghamMedicalSchool,Queen’sMedicalCentre,NottinghamNG72UH,UK}

c_{TheRowettInstituteofNutritionandHealth,UniversityofAberdeen,Bucksburn,AberdeenAB219SB,UK}

h

i

g

h

l

i

g

h

t

s

•Theviral2AsequenceissuitableforgenemanipulationintheSiberianhamster. •Itallowslong-termsimultaneousover-expressionof2genesinvitroandinvivo. •WedemonstratedualexpressioninvitrointheneuroblastomacelllineSH-SY5Y. •WedemonstratedualexpressioninthehypothalamiofSiberianhamstersandmice.

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received22May2015

Receivedinrevisedform22July2015 Accepted12August2015

Availableonline20August2015

Keywords: Viral2Asequence

Recombinantadeno-associatedvirus (rAAV)

NeuroblastomaSH-SY5Ycells VGF(non-acronymic)

Enhancedgreenfluorescentprotein(eGFP) Siberianhamster

a

b

s

t

r

a

c

t

Introduction:Theviral2Asequencehasbecomeanattractivealternativetothetraditional internal ribosomalentrysite(IRES)forsimultaneousover-expressionoftwogenesandincombinationwith recombinantadeno-associatedviruses(rAAV)hasbeenusedtomanipulategeneexpressioninvitro.

Newmethod:TodeveloparAAVconstructincombinationwiththeviral2Asequencetoallowlong-term over-expressionofthevgfgeneandfluorescentmarkergenefortrackingofthetransfectedneurones

invivo.

Results:TransienttransfectionoftheAAVplasmidcontainingthevgfgene,viral2AsequenceandeGFPinto SH-SY5YcellsresultedineGFPfluorescencecomparabletoacommerciallyavailablereporterconstruct. ThisincreaseinfluorescentcellswasaccompaniedbyanincreaseinVGFmRNAexpression.Infusion oftherAAVvectorcontainingthevgfgene,viral2AsequenceandeGFPresultedineGFPfluorescence inthehypothalamusofbothmiceandSiberianhamsters,32weekspostinfusion.Insituhybridisation confirmedthatthelocationofVGFmRNAexpressioninthehypothalamuscorrespondedtotheeGFP patternoffluorescence.

Comparisonwitholdmethod:Theviral2AsequenceismuchsmallerthanthetraditionalIRESandtherefore allowedover-expressionofthevgfgenewithfluorescenttrackingwithoutcompromisingviralcapacity.

Conclusion:Theuseoftheviral2AsequenceintheAAVplasmidallowedthesimultaneousexpression ofbothgenesinvitro.WhenusedincombinationwithrAAVitresultedinlong-termover-expressionof bothgenesatequivalentlocationsinthehypothalamusofbothSiberianhamstersandmice,withoutany adverseeffects.

©2015TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense (http://creativecommons.org/licenses/by/4.0/).

Abbreviations:Allstandardinthisfield

∗Correspondingauthor.Tel.:+4401159516604;fax:+4401159512212. E-mailaddress:[email protected](P.H.Jethwa).

1. Introduction

Theuseoftransgenicandknockoutmicehascontributedgreatly toourunderstandingofgenefunctionanddisease.However,these micemodelsarenotonlycomplextocreate,butmaynotbeviable, ormayproducea complexphenotypereflectingdevelopmental orfunctionalcompensation.Genetransfertechnologiesutilising

http://dx.doi.org/10.1016/j.jneumeth.2015.08.013

J.E.Lewisetal./JournalofNeuroscienceMethods256(2015)22–29 23

virusesprovideameanstoovercomesomeoftheselimitations.The mostwidelyusedviralvectorsforneuronalover-expressionare recombinantadeno-associatedviruses(rAAV),astheyhavebeen showntoefficientlyandstably transducea varietyoftissuesin immunocompetentanimals,includingmice,ratsandSiberian ham-sters(DayaandBerns,2008;Jethwaetal.,2010).Howeverthese virusesarelimitedintheircapacitytoincorporateforeignDNAby virtueoftheirgenomicsize(approximately5kb)(Kayetal.,2001). Althoughhybridswithotherviruseshavebeengeneratedtotryto overcomethislimitation(Costantinietal.,1999;Nakaietal.,2000; Recchiaetal.,1999),thelowinsertcapacityremainsanissueif co-expressionofgenesisrequiredfromasingleviralvector.For example,thecombinationofageneofinterestandareportergene, suchasgreenfluorescentprotein(GFP),is particularlyusefulin ordertoenablevisualisationoftransducedcellsbothinvitroand

invivo.

Co-expression of genes can greatly enhance the efficiency and usefulnessof transgenic applications.Anumber of options areavailable tosimultaneouslyexpresstwogenes inparticular cells/neurones. Currently themostcommon approach relies on vectorstrategies inwhichgenesare linkedby aninternal ribo-somalentrysite(IRES)toallowsimultaneousexpressionofgenes, sincetheIRESsequencepermitstheproductionofmultiple pro-teinsfromasinglemRNAtranscript.RibosomesbindtotheIRESin a5-capindependentmannerandinitiatetranslation(Jangetal., 1988).However,therearetwomainlimitationstotheuseofthe IRES.First,thesizeoftheIRESisofteninexcessof500basepairs, whichfurtherlimitsthecapacitytoincorporateforeignDNA. Sec-ond,theexpressionofthedownstreamgenewithinthevectoris oftenreducedand thereforeexpressionofthetwo genesisnot equivalent.Recentlyithasbeenshown(Chanetal.,2011)thatviral 2Asequencescanovercometheselimitations;withtheviral2A sequenceshowntoincreasereportergeneexpressionin compari-sontotheIRESsequence.Thustheviral2Apeptidesequenceoffers analternativetotheIRES.

Theviral2Asequencewasfirstidentifiedinthepicornaviruses (Ryan et al., 1991; Trichas et al., 2008). Viral 2A sequences arerelativelyshort(approximately20 aminoacids)andcontain theconsensusmotifAsp-Val/Ile-Glu-X-Asn-Pro-Gly-Pro(Ibrahimi etal.,2009).Thesequenceactsco-translationally,theformationof anormalpeptidebondbetweentheglycineandprolineresidues isprevented,whichresultsinribosomalskipping(Donnellyetal., 2001) and therefore cleavage of the nascent polypeptide. This effectproducesmultiplegenesatequimolarlevels.Theefficiency oftheviral2Asequencehasbeendemonstratedinawiderange ofeukaryoticcells fromyeasttomammals(deFelipe andRyan, 2004),includingitseffectivenessfollowinginsertionbetweentwo reportergenesinvitro(RyanandDrew, 1994).Importantly,the viral2A sequence,in combination withAAV,wasableto func-tionintheratbrainwithoutanycytotoxiceffects(Furleretal.,

2001).

Despitetheseadvantages,theuseofviral2Asequence technol-ogyforinvivoresearchhasbeenlimited.Inthisstudy,weutilised the viral2A sequence (a) toovercome AAV capacity problems facedutilisinglargegenesand(b)toover-expressVGFandeGFP inthehypothalamusofSiberianhamstersandmicetodetermine itsfeasibilityinstandardandnon-standardlaboratoryanimals.We clearlydemonstratethatthecombinationofAAVwiththeviral2A

Table1

PCRprimersusedforthesynthesisofVGF-2Afragment,todetermineendogenous VGFmRNAandcyclophilincontrol.

Gene Forwardprimer(5–3) Reverseprimer(5–3)

ModifiedPCRprimers (foramplificationof VGFcDNA)

CCCGGGAAGCTTACC ATGAAAACCTTCACG TTGCCGGCATCC

GGGCCC

TGGGCCAGGATTCTC CTCGACGTCACCGCA TGTTAGCAGACTTCC TCTGCCCTCTCCACT GCCGAGCACGTGCTG CTGCACCGCCCG

VGFmRNA GACCCTCCTCTCCAC

CTCTC

ACCGGCTCTTTATGC TCAGA

CyclophilinA TCCTGCTTTCAAGAA TTATTCC

ATTCGAGTTGTCACA GTCAGC

ThemodifiedPCRprimers fortheamplificationofVGFcDNA(Accessionno.: NM001039385.1)fromthepSC-B-AMP/KANbluntcloningvectortoproducethe VGF-2Afragment.TheforwardprimercontainsaKozaksequence(inbold)for initiationoftranslation,whilethereverseprimercontainstheviral2Asequence (underlined)andapointmutationtoallowtheremovalofthestopcodonfromthe VGFcDNA.Bothprimerscontaineduniquerestrictionsitestoaidthesub-cloning process.TheVGFmRNAandcyclophilinprimersweredesignedfromdatabase sequences.SequencedatawasinputintoPrimer3inFASTAformatandprimers weredesignedusingthedefaultcriteria(Primersize;18–27bp,Meltingtemperature 55–62◦_{C,Ampliconsize50–150bp)andobtainedfromSigma,Dorset,UK.}

sequenceresultsinlongtermover-expressionofVGFmRNAand theeGFPreportergeneinthehypothalamusofbothspecies.

2. Materialsandmethods

2.1. Synthesisofconstructandviralparticles

ConstructionofthepAAV-CBA-AgRP-IRES-eGFP-WPREplasmid (fromanoriginalplasmidAAVvector,akindgiftfromDrMiguel Sena Esteves,University of Massachusetts,Worcester, USA) has beendescribedpreviously(Jethwaetal.,2010).Wereplacedthe AgRP-IRESinthisplasmidwithVGFcDNAandtheviral2Asequence fromTrichasetal. (2008)(Fig.1).FulllengthmouseVGFcDNA (Accessionno.:NM001039385.1)wasisolatedandinsertedinto thebluntcloningvector,pSC-B-AMP/KAN(AgilentTechnologies, UK)for amplificationusing modified PCR primers.The forward primer containedaKozaksequencefor initiationoftranslation, whilethereverseprimercontainedtheviral2Asequenceanda pointmutationtoallowtheremovalofthestopcodonfromthe VGFcDNA(Table1).

Theresulting PCRfragment,which containedtheVGF cDNA and viral2A sequence(VGF-2A), wasdigestedwithHindIIIand

NotI,whiletheAgRP-IRESsequencewasexcisedfromthe pAAV-CBA-AgRP-IRES-eGFP-WPREplasmidvectorusingHindIIIandAgeI, thelatterproducinganidentical5overhang(5-GGCC-3)toNotI. The digested plasmid was treated with calf intestinal alkaline phosphatase(Promega,USA)topreventre-ligationoftheplasmid withoutinsert.Sure2Supercompetentcells(AgilentTechnologies, USA) were used for transformation toprevent DNA rearrange-ment/deletion,astheylacktheEscherichiacoligenesimplicatedin suchevents,therebyimprovingcloningefficiency.TheVGF-2APCR fragmentproducedbyPCRwasclonedintotheoriginalAgRP plas-midtoproducethepAAV-CBA-VGF-2A-eGFP-WPREplasmidvector (referredtoaspAAV-VGF-GFP)(Fig.2).

ggc agt gga gag ggc aga gga agtctg cta aca tgc ggt gac gtc gag gag aat cct ggc cca Gly Ser Gly Glu Gly Arg Gly Ser LeuLeu ThrCys Gly Asp Val Glu Glu AsnPro GlyPro

VGF

2A

GFP

Fig.2. AschematictoshowstepsleadingtotheproductionofrAAV-VGF.FulllengthmousecDNAwasisolatedandinsertedintothebluntcloningvectorpSC-B-AMP/KANfor amplificationusingmodifiedprimerstoproducetheVGF-2Afragment.ThisproductwasclonedintothepAAV-CBA-AgRP-IRES-GFPbackbone(previouslyusedinJethwaetal., 2010),followingexcisionoftheAgRP-IREScomplextoproducepAAV-CBA-VGF-2a-GFP(pAAV-VGF-GFP).Thiswasusedfortheinvitrochecking(Study1),beforethenbeing usedtoproducerecombinantAAV-2(rAAV)byVectorBioLabs(USA)yieldingrAAV-VGFforinvivoinfusion(study2).ITR—internaltandemrepeat,CMV/IE—cytomegalovirus intermediateearlypromoter,CBA—chicken␤-actinpromoter,eGFP—enhancedgreenfluorescentprotein,WPRE—WoodchuckHepatitisviruspost-transcriptionalregulatory element.

Following in vitro validation in SH-SY5Y cells, the pAAV-VGF-GFPplasmidwaspackagedinto AAV-2particles byVector BioLabs(PA, USA). The titreobtained was 7.2×1012_{gc/ml and}

will be referred to as rAAV-VGF. The rAAV-GFP control vector waspurchased from VectorBiolabs (PA, USA);this had a viral titreof1×1013_{gc/mlandexpressedeGFPunderthecontrolofa}

cytomegalovirus(CMV)promoter.

2.2. TheSH-SY5Yneuroblastomacellline

ThehumanneuroblastomaSH-SY5Ycellsweregrownin25cm2

tissue cultureflasks withcomplete Dulbecco’s modified Eagle’s medium/nutrientmixtureF-12Ham,containing10%foetalbovine serum,100units/lpenicillinand100mg/lstreptomycin(all Sigma-Aldrich, Dorset, UK), which will be referred to as DMEM/F12 complete.Culturesweremaintainedat37◦Cina95%humidified incubatorwith5%CO2.Cellswereroutinelysplit1:3with0.05%

trypsin(Sigma-Aldrich,Dorset,UK)every48h.Fordifferentiation, theSH-SY5Ycellswereseededat5×104_cells/cm2_in6-well

tis-suecultureplates(9.5cm2_{)inDMEM/F12complete,and48hlater}

treatedwith10␮Mretinoicacid(RA)(Sigma-Aldrich,Dorset,UK) for120hinaccordancewithpreviousstudies(Cheungetal.,2009; Pahlmanetal.,1984).CellswerevisualisedbyLeicaDF420C micro-scope(LeicaInstruments,MiltonKeynes,UK).

2.3. Study1:InvitroassessmentofviralplasmidpAAV-VGF-GFP

2.3.1. Transfection

pAAV-VGF-GFPandpZsGreen1-N1(apositivecontrol;Clontech Laboratories,CA,USA) plasmidswere transfectedinto undiffer-entiated SH-SY5Ycells using FuGENEHD (Promega,Wisconsin, USA) according to the manufacturer’s protocol. Briefly, prior to transfection, cells at approximately 80% confluence were

transferredto OptiMeM1® _{reduced serummediacontaining no}

antibiotics(Promega,Wisconsin,USA).PlasmidDNAwasdiluted inOptiMEM1®_{togiveaconcentrationof20␮g/ml.TheFuGENE}®

HDtransfectionreagentwasaddedtoachievea3:1,reagent:DNA ratioand 10␮lofthereagent:DNA mixturewasadded toeach wellequating to50ngofplasmidDNAperwell.Thecellswere incubatedfor72hpriortoassessmentofeGFPfluorescenceand VGFmRNAexpression.Cellstransfectedinparallelwerefurther incubatedwithRA(10␮M)toinducedifferentiationpriortoeGFP assessment.

2.3.2. InvitroeGFPexpression

eGFP fluorescence in the SH-SY5Y cells was imaged using the Typhoon Trio+ instrument (excitation maximum=493nm; emissionmaximum=505nm)andImageQuantsoftware(GE Life-Sciences,UK).

2.3.3. InvitroVGFmRNAexpression

2.3.3.1. RNAextraction. Prior toharvestingthecells, theDMEM completemediumwasremoved,thencellswerescrapeddirectly into200␮lof RNase-freephosphatebufferedsaline(PBS).Total RNAwasextractedfromthecellsusingtheHighPureIsolationkit accordingtothemanufacturer’sinstructions(RocheLifeScience, WestSussex,UK)andasdescribedpreviously(Brownetal.,2012).

J.E.Lewisetal./JournalofNeuroscienceMethods256(2015)22–29 25

RNaseinhibitor(20U)and4␮lreactionbufferwereaddedtoeach reactionon ice,vortexed and incubatedfor 10min at25◦C for 10min,followedbyincubationsat55◦C(30min),85◦C(5min)and then4◦Ctocool.ThecDNAwasstoredat20◦C.

2.3.3.3. QuantitativeRT-PCR. QuantitativePolymeraseChain Reac-tions(PCR)wereperformedwithSYBR®_{greenoptimisedforthe}

LightCycler480(RocheLifeScience,UK).Allreactionswere per-formed in triplicate on 384 well plates as per manufacturer’s instructions.Brieflyeachwellcontained5␮lcDNAinSYBR®

Mas-terMix1,0.25␮Mprimers,andRNasefreewaterinatotalvolume of15␮lperwell.Sampleswerepre-incubatedat95◦Cfor5min, followedby40amplificationcycles(de-naturation:95◦Cfor10s; annealing:55◦Cfor10sandelongation:72◦Cfor30s).The respec-tiveprimersetsusedcanbeseeninTable1.

Ctvalueswereconvertedtoexpressionlevelsusingastandard

curveproducedusingaserialdilutionofapooledcDNAmadefrom allsamplestochecklinearityandefficiencyofthePCRreactions. ExpressionvalueswerethennormalisedtocyclophilinA,themost stablereferencegeneundertheexperimentalconditions.

2.4. Study2:InvivoassessmentoftherAAV-VGFviralconstruct

2.4.1. Animals

AllanimalprocedureswereapprovedbytheUniversityof Not-tinghamLocalEthicalReviewCommitteeandwerecarriedoutin accordancewiththeUKAnimals(ScientificProcedures)Act1986 (projectlicencePPL40/3604).

MaleSiberianhamsters(Phodopussungorus)aged4–6months (n=12)weretakenfromacolonymaintainedbytheUniversityof Nottingham(Ebling,1994).Theywerehousedinindividualcages undercontrolledtemperature(21±1◦C)andona photocycleof 16hlight/8hdark(lightsoffat11:00h),withadlibitumaccessto foodandwater,unlessotherwisestated.Thedietwasstandard lab-oratorychowcomprisingof19%extrudedproteinand9%fat(Teklad 2019,Harlan,UK).

Male C57BL/6Jmice(Mus musculus)aged 3months (n=12), were supplied by Harlan, UK. They were housed in individual cagesundercontrolledtemperature(21±1◦C)and ona photo-cycleof12hlight/12hdark(lightsoffat19:00),withadlibitum

accesstofoodandwater.Thedietwasstandardlaboratorychow comprising of18% extruded proteinand 6.2% fat(Teklad2018, Harlan,UK).

2.4.2. Infusionofviralvectors

Animal surgical procedures were carried out as previously described(Jethwaetal.,2010).Briefly,animalswereplacedina Kopfstereotaxicframe(DavidKopfInstruments,NY,USA)under generalanaesthesia(0.5–2.5%isoflurane),withtheincisorbar pos-itionedlevelwiththeinterauralline.Usingthesuturesconfluence bregmaasalandmark,asmallholewasdrilledonmidline.The duramaterwaspiercedjustlateraltothemid-sagittalsinusand a drawn glass capillary microinjector (30-micron tip diameter) wasloweredtothecorrectlocation.Usinga NanolitreInjection system(WPI,Stevenage,UK), Siberianhamsters(n=6/group)or mice(n=6/group)werebilaterallyinfusedwith200nloftheviral vector (rAAV-GFPor rAAV-VGF-GFP) directed towardsthe PVN (anteroposterior +0.03, mediolateral ±0.03, dorsoventral −0.58 (co-ordinatesfromPaxinosandFranklin,2012)overoneminute. Followinginfusion,theglassmicroinjectorwaskeptinplaceforan additional5mintoallowfordiffusionandpreventionofbackflow throughthecannulatrack,theincisionwasclosedusingMichel clips. Analgesia was maintained via subcutaneous injection of carprofen(50mg/kgRimadyl,Pfizer,Kent,UK)administeredbefore surgery.Thesurgically-preparedSiberianhamstersandmicewere alloweda seven dayrecovery period, during which theywere

handledonadailybasis,receivedanalgesiaandhadaccesstoa palatabledietconsistingofsoakedTeklabdiet.Oneweekpostviral infusion,Siberianhamstersandmiceunderwentmetabolicanalysis to determinelocomotor activity in the Comprehensive Labora-toryAnimalMonitoringSystem(CLAMS)aspreviouslydescribed (Jethwaet al., 2010).At 32 weeks postinfusion, animals were euthanisedbyinjectionofpentobarbitalsodium(Euthatal;Rhone Merieux, Harlow, UK). Brains were immediately removed and snapfrozenondry iceforsubsequentmeasurementofGFPand VGFexpressionbyfluorescenceimagingandinsituhybridisation, respectively.

2.4.3. Insituhybridisation

Coronal sections (20␮m) of brains were cut, using a cryo-stat(Microm,USA),andarrangedon3-aminopropyltriethoxysaline (APES) coated slides. Sections on the slides were fixed in 4% paraformaldehyde(PFA)beforebeingdriedforstorageat−70◦C. Riboprobes were generated from the blunt pSC-B-AMP/KAN plasmid (containing VGF cDNA)and pSLAX13-eGFP (containing eGFP—akindgiftfromDrDylanSweetman,Universityof Notting-ham).AntisensetranscriptsweregeneratedusingT7polymerase (NEB,USA) inthepresenceofdigoxigenin (DIG)/fluorescein-12-uridine-5-triphosphate, which could subsequently be detected. Fluorescence in situ hybridisation was performed as described previously(Sweetmanetal.,2008).Briefly,riboprobeswere puri-fiedona G-50spincolumn(BoehringerMannheim,USA) anda smallaliquotsubjectedtogelelectrophoresistodetermineyield and integrity of the riboprobes. Slides containing the sections werethawed toroomtemperature,fixedwithfreshlyprepared 4%PFA/0.1%gluteraldehydebeforebeingtreatedwithproteinase K(10␮g/ml).Slideswereincubatedwith80␮l1×hybridisation solution(Sigma,St.Louis,MO,USA)containing1␮lofriboprobe for6hat65◦C/minatroomtemperature.Acoverslipwasplaced on each slide and slides were incubated overnightat 65◦C in a humidified in situ hybridisation chamber. Post hybridisation, the section were washed twice with hybridisation solution at 65◦C for10min,followedby 2washes withmaleicacidbuffer containing 0.1% Tween-20 (MABT, pH 7.5) at room tempera-ture. The sections were blocked with MABT/2%Roche blocking agent (Roche,USA) for 1hat roomtemperature andincubated overnightwithanti-digoxigeninantibodyconjugatedtoalkaline phosphatase(1:2000)at4◦C.Slidesweresubsequentlywashed3×

withMABTfor1hatroomtemperaturefollowing anovernight incubation at 4◦C in MABT. For the colour reaction, sections were washed with1-methyl-5-thiotetrazole (NMTT) containing nitroblue tetrazolium (NBT) and 5-bromo-3-indoxyl-phosphate (BCIP), which yields a black–purple precipitation. The reaction wasstoppedbywashingthesectionsin5×TBSTw(1×TBS,0.1% TweenTM _{20, 0.2mMsodiumazide)overnightat 4}◦_{C. This}

pro-cesswasrepeatedthefollowing daytointensifythesignaland reducebackground.Cover slipswereappliedtotheslides with asmallamountofVectashieldmedia(VectorLabs,CA,USA)and sealedwithnailpolish.ImageswerecapturedusingaLeicaDMRB microscope (Leica Microsystems, Germany) and Openlab soft-ware(UK). eGFP wasexcitedusing the488nm laser (emission 520nm).

2.5. Statisticalanalysis

Fig.3. eGFPandVGFexpressioninSH-SY5YcellsfollowingtransienttransfectionwiththepAAV-VGF-GFPplasmid.(A)eGFPexpressioninSH-SY5Ycells72hposttransfection withthepAAV-VGF-GFPplasmid.(B)eGFPexpressionintheSH-SY5YcellstransfectedwiththepAAV-VGF-GFPplasmidfollowingdifferentiationwith10␮MRAfor120h. Quantificationof(C)eGFPexpressionand(D)VGFmRNA72hposttransfectionwithpAAV-VGF-GFPorpZsGreen1-N1(control)inundifferentiatedSH-SY5Ycells.Valuesare groupmeans±SEM(n=3),***p<0.0001vs.control.

3. Results

3.1. Study1:InvitroassessmentofviralplasmidpAAV-VGF-GFP

Prior to viral packaging, the ability of the pAAV-VGF-GFP plasmid to simultaneously express both VGF and eGFP was verifiedfollowing transienttransfection of SH-SY5Ycells. Fluo-rescentmicroscopyconfirmedwidespreadexpressionofeGFPin undifferentiatedSH-SY5Y cells 72h posttransfection (Fig. 3A). TreatmentofthetransfectedSH-SY5Ycellswith10␮MRAfor120h induceddifferentiation,atwhichpointeGFPexpressionexpanded beyondthe cell body and extended to the neurite projections (see Fig. 3B). The increase in eGFPexpression in undifferenti-ated cells was similar to that observed following transfection of the SH-SY5Y cell line with the positive control construct, pZsGreen1-N1,expressingZsGreen1-N1underthecontrolofaCMV promoter(GFPexpression:pZsGreen1-N1;442.8±8.8, pAAV-VGF-GFP;366.0±6.5,p=n.s.Fig.3C).Furthermore,theincreaseineGFP expressioninundifferentiatedcellswasassociatedwithanincrease inVGFmRNAexpression.TransfectionoftheSH-SY5Ycellswith pAAV-VGF-GFPresultedina20-foldincreaseinVGFmRNA expres-sion(VGFmRNA:20.6±0.1,F=5.830,p<0.0001,Fig.3D),compared tocontrolcellstransfectedwithpZsGreen1-N1.

3.2. Study2:InvivoassessmentofviralconstructrAAV-VGF

3.2.1. Animalbehaviour

Approximately95% of theanimals that underwent theviral microinjectionsurgeryrecoveredfullyfromanaesthesiaandwere reintroducedtotheirhomecages.Oncereintroduced,allanimals maintainedgoodhealththroughoutthedurationofthestudyand normalbehaviourwasobserved,withnochangesininitialfood intakeorlocomotoractivityobservedoneweekpostviralinfusion (Table2).

3.2.2. HypothalamicexpressionofeGFPandVGFmRNA

Post mortem analysis of eGFP by fluorescence microscopy wasperformedat32weekspostinfusionoftheviralconstructs

Table2

Comparisonoffoodintakeandlocomotoractivityoneweekpostviralinfusionin theSiberianhamsterandmouse.

Foodintake(g) Locomotoractivity (beambreaks)

rAAV-GFP rAAV-VGF rAAV-GFP rAAV-VGF

Siberian hamster

3.71±0.24 4.01±0.07 23.10±5.10 23.11±7.38

Mouse 3.76±0.32 4.13±0.25 97.93±9.17 100.13±17.16

Meanfoodintake(g)measuredinthehomecageandmean24hvaluesforthe num-berofbeambreaksinthemetaboliccages(ComprehensiveLabAnimalMonitoring System;CLAMS).Valuesaremean±SEM.

rAAV-GFPandrAAV-VGFinSiberianhamstersandmice. Expres-sionofeGFPwaswidespreadinthehypothalamusofbothspecies, aswellasalongtheinfusiontract.eGFPexpressionwasobserved inbothrAAV-GFPand rAAV-VGFinfusedSiberianhamstersand mice.ThespreadoftheeGFPexpressionfromviralconstructswas similarforbothrAAV-GFPandrAAV-VGFvectors,upto400␮min thelateralplane(Fig.4).Asseeninpreviousstudies(Jethwaetal., 2010),therewereinstancesofunilateralexpressioninseveralof theanimals,whichwasindependentofthevectorinfused(Fig.4). Withinthehypothalamus,eGFPexpressionwasapparentalong the third ventricle, suprachiasmatic nucleus, lateral hypothala-micarea,theanteriorhypothalamicnucleus,theparaventricular nucleus(PVN),arcuatenucleus(ARC),ventromedialhypothalamus anddorsomedialnucleusinbothspecies receivingeither rAAV-GFPorrAAV-VGFviralvectors(Fig.4).Confocalanalysisrevealed therewaswidespreadeGFPexpressioninthecellsomaandcell processesofthePVNofbothSiberianhamsters(Fig.4CandG)and mice(Fig.4DandH)receivingeitherrAAV-GFPorrAAV-VGFviral vectors.

J.E.Lewisetal./JournalofNeuroscienceMethods256(2015)22–29 27

Fig.4.Useoftheviral2AsequenceintherAAV-VGFconstructresultsinwidespreadeGFPexpressioninthehypothalamus.RepresentativepicturesofeGFPexpressioninthe hypothalamusofSiberianhamstersandmicewhichreceivedbilateral(200nl)injectionsofeitherrAAV-GFPorrAAV-VGFdirectedtothePVN.(A–D)eGFPexpression followinginfusionofrAAV-GFPcontrolvectorinthewholehypothalamusof(A)Siberianhamstersand(B)miceandinthecellsomaandprocessesofthePVNof(C)Siberian hamstersand(D)mice.(E–G)eGFPexpressionfollowinginfusionoftherAAV-VGFvectorinthehypothalamusof(E)Siberianhamstersand(F)miceandinthecellsomaand processesofthePVNof(G)Siberianhamstersand(H)mice.Scalebar=2.5mm.

VGFmRNAinlowamountsintheARC(Fig.5),indicatingareasof endogenousVGFexpression.

4. Discussion

Theabilitytoco-expresstwoormoregenesinthesamecellsis highlydesirable,particularlyforidentificationoftransfectedcells followingviraltransfectioninvivo.Wehaveshownthattheviral 2AsequenceallowsthesimultaneousexpressionofbothGFPand thegene ofinterest(VGF)inthebrainsofanon-standard labo-ratoryspecies,theSiberianhamster,whichiscomparabletothe mouse.

TheroleofVGFwasfirsthighlightedbythephenotypeofmice lacking the functional vgf (non-acronymic) gene (Hahm et al., 1999),whichsuggestedananabolicroleforthisgene.Howeverwe havepreviouslyshownthatVGFmRNAexpressionintheSiberian hamsterisregulatedinrelationtochangesinitsbodyweightcycle, withsignificantlyhigher hypothalamicexpressioninthewinter catabolicstateascomparedtothesummeranabolicstate(Barrett etal.,2005).SubsequentfunctionalstudiesusingVGF-derived pep-tidesinbothmiceandSiberianhamsters,havesuggestedacatabolic role(Bartolomuccietal.,2006;Jethwaet al.,2007;Lewisetal., 2015).Thelackofantibodiestowardsthisgeneforthedetection ofspecificderivedpeptides,aswellasouruseofanon-standard animal model,directed us todevelop a novelstrategyto over-expressVGFinthehypothalamusofadultSiberianhamsters.We alsowantedtovisualisetransfectedcellsusingareportergene,as

previouslyobservedinourstudiesontheover-expressionofAgRP (Jethwaetal.,2010).

Whileourpreviousstudieshaveshownthatover-expression oftwogenesusingIRESisfeasible(Jethwaetal.,2010),thesize oftheIRESandVGFgenealonewouldhaveaccededthecapacity oftherAAVandpossibleleadingtoissueswithregardsto suffi-cienttitre.Thuswedesignedavectorutilisingtheviral2Asequence forsimultaneousexpressionofVGFmRNAandeGFP.Theviral2A sequencehasnotonlybeenshowntobereliable,butalsocauses nocytotoxiceffectsintransgenicmice(Trichasetal.,2008).The viral2Asequenceisparticularlyusefulinvectorstrategies(AAV andretroviralvectors)wherethecapacitytoincorporateforeign DNAislimited.Indeed,theviral2Asequencehasbeenshownto reducethesizeoftheviralvector,improvingtransfectionefficiency andviraltitre,andthustransductionefficacy,leadingtoincreased expressionofboththegeneofinterestandreporter(Szymczakand Vignali,2005).

Fig.5.VGFmRNAandeGFPexpressionpatternsaresimilarfollowingrAAV-VGFinfusionintothehypothalamusofbothSiberianhamstersandmice.Representativepicturesof eGFPexpression(left)andVGFmRNAexpressionviainsituhybridisation(right)inthehypothalamusofSiberianhamsters(AandB)andmice(CandD)receivingbilateral injectionsofeitherrAAV-GFP(AandC)orrAAV-VGF(BandD)directedtothePVN.Scalebar=2.5mm.

resultedinexpressionofeGFPbeyondthecellbodyintotheneurite projections.

TheAAVvector(rAAV-VGF)wasthenproducedfromthis plas-midvectorandinfusedintothehypothalamusofSiberianhamsters andmice toconfirmtheeffectiveness ofthe viral2A sequence

invivo.Wehaveshownthattheover-expressionwasstillpresentin bothspeciesat32weekspostmicroinjection,whileinsitu hybrid-isationshowedthattheVGFmRNAexpressionwasco-localised witheGFPexpression.Whetherthisover-expression isequal in bothspeciesremainstobedetermined;unfortunatelytheinsitu

hybridisationmethodusingtheDIGdoesnotlenditselfto quantifi-cation,asthecolourreactionisrepeatedtoincreasetheintensityof thesignalandreducethebackground.Thismayaccountforthe dif-ferencesbetweenthespecies,assectionswereanalysedinbatches accordingtospeciesratherthantreatment.

Nonetheless, the function of viral2A sequence appears not toshowanyattenuationinexpressionlevelsacrossgenerations (Trichasetal.,2008).Postmortemanalysisrevealedahighnumber ofcells,overalargeareaofthebrain,expressedthevectorsat32 weekspostinfusion.Despitethebilateralinfusionof200nlanda micropipettetipof30_␮m,itwasapparentthatcells300–400_␮m awayalsoexpressedthevector.Cellsmoredorsalinthebrain adja-centtothetractofthemicropipettealsoexpressedthevectors. Higherlateralexpressionwasnotedinsomeoftheanimals,a reflec-tionofatechnicalissuewiththeinfusionofviralvectorsatlow volumesthroughsmalldiameters,somethingwehavenoted pre-viously(Jethwaetal.,2010).Furthermorethismayresultinareas ofunevenexpressionpotentiallygivingrisetodifferencesbetween speciesand/oranimals.

Thus,wehaveshownthattheviral2Asequencecanbeused successfullytoover-expresstwogenessimultaneouslyinvitroand

invivo.Indeed,Trichasetal.(2008)statedthattheadvantageof using2AsequencesoverthecommonlyusedIRESsequenceisthe

abilitytoco-expressbothgenesatnearlyequallevels.Indeed10 fold differences in IRES-controlled expression have been noted (Martinetal.,2006),whilethelengthofthegenepreceding(5)the IREShasrecentlybeenshowntoinfluencetheexpressionofthe fol-lowing(3totheIRES)reporter(Payneetal.,2013),demonstrating theunreliabilityofthesequence forsimultaneous gene expres-sion. These effects have not been observed for plasmidand/or viralvectorswhichutilisetheviral2Asequence,whichis partic-ularlyimportantwhenhighlevelsofexpressionofbothgenesare required(Furleretal.,2001).However,aswithallsystems,thereis apotentiallimitationtotheuseoftheviral2Asequence.Sincethe viral2AsequenceisaddedtotheC-terminaloftheproteinencoded upstreamofthesequence,thismayaffectfunctionand/oractivityof thatprotein,althoughthishasnotbeenobservedinanyofthe stud-iespublishedtodate(Furleretal.,2001;Trichasetal.,2008).Further studiesconcerningthefunctionandsafetyoftheviral2Asequence arerequired,butnoadverseeffectswerenotedinourstudies fol-lowinginfusionofviral2Asequencecontainingvectors.Indeed, thesequencesuccessfullymediatedco-translational‘cleavage’to expresstwogeneswithinthehypothalamusofSiberianhamsters akintothatinthemouseandotherrodentmodels(Furleretal., 2001;Trichasetal.,2008).

Overall,theresultsfurthersupporttheuseofAAVvectorsfor manipulatinggene functioninvivo,particularlyinnon-standard laboratorymodels.WehaveshownthattheAAVconstructusing theviral2AsequenceresultedinsimultaneousexpressionofVGF andeGFP,bothinvitroandinvivo.Furthermore,theAAVconstruct resultsinlong-termexpressioninvivo.

Acknowledgements

J.E.Lewisetal./JournalofNeuroscienceMethods256(2015)22–29 29

TheworkwassupportedbyUniversityofNottinghamKnowledge TransferAwardandtheBBSRCStrategicSkillsAward.

References

BarrettP,RossAW,BalikA,LittlewoodPA,MercerJG,MoarKM,etal. Photope-riodicregulationofhistamineH3receptorandVGFmessengerribonucleic acidinthearcuatenucleusoftheSiberianhamster.Endocrinology2005;146: 1930–9.

BartolomucciA,LaCorteG,PossentiR,LocatelliV,RigamontiAE,TorselloA,etal. TLQP-21,aVGF-derivedpeptide,increasesenergyexpenditureandprevents theearlyphaseofdiet-inducedobesity.ProcNatlAcadSciUSA2006;103: 14584–9.

BrownDM,ParrT,BrameldJM.MyosinheavychainmRNAisoformsareexpressed intwodistinctcohortsduring C2C12myogenesis.JMuscleResCell Motil 2012;32:383–90.

ChanHY,VS,XingX,KrausP,YapSP,NgP,etal.ComparisonofIRESand F2A-basedlocus-specificmulticistronicexpressioninstablemouselines.PLoSONE 2011;6:e28885.

CheungYT,LauWK,YuMS,LaiCS,YeungSC,SoKF,etal.Effectsofall-trans-retinoic acidonhumanSH-SY5Yneuroblastomaasinvitromodelinneurotoxicity research.Neurotoxicology2009;30:127–35.

CostantiniLC,JacobyDR,WangS,FraefelC,BreakefieldXO,IsacsonO.Genetransfer tothenigrostriatalsystembyhybridherpessimplexvirus/adeno-associated virusampliconvectors.HumGeneTher1999;10:2481–94.

DayaS,BernsKI.Genetherapyusingadeno-associatedvirusvectors.ClinMicrobiol Rev2008;21:583–93.

deFelipeP,RyanMD.Targetingofproteinsderivedfromself-processing polypro-teinscontainingmultiplesignalsequences.Traffic2004;5:616–26.

Donnelly ML, Hughes LE, Luke G, Mendoza H, ten Dam E, Gani D, et al. The‘cleavage’ activities of foot-and-mouth disease virus 2A site-directed mutantsandnaturally occurring‘2A-like’sequences. JGenVirol 2001;82: 1027–41.

EblingFJP. Photoperiodicdifferencesduring developmentinthe Dwarf Ham-sters Phodopus-sungorus and Phodopus-campbelli. Gen Comp Endocrinol 1994;95:475–82.

FurlerS,PaternaJC,WeibelM,BuelerH.RecombinantAAVvectorscontaining thefoot andmouthdiseasevirus2A sequenceconferefficient bicistronic geneexpressioninculturedcellsandratsubstantianigraneurons.GeneTher 2001;8:864–73.

HahmS,MizunoTM,WuTJ,WisorJP,PriestCA,KozakCA,etal.Targeteddeletion oftheVgfgeneindicatesthattheencodedsecretorypeptideprecursorplaysa novelroleintheregulationofenergybalance.Neuron1999;23:537–48.

IbrahimiA,VandeVeldeG,ReumersV,ToelenJ,ThiryI,VandeputteC,etal.Highly efficientmulticistroniclentiviralvectorswithpeptide2Asequences.HumGene Ther2009;20:845–60.

JangSK, KrausslichHG,NicklinMJ,Duke GM,PalmenbergAC,Wimmer E.A segmentofthe5nontranslatedregionofencephalomyocarditisvirusRNA directsinternalentryofribosomesduringinvitrotranslation.JVirol1988;62: 2636–43.

JethwaPH,WarnerA,FowlerMJ,MurphyM,deBackerMW,AdanRA,etal. Short-daysinduceweightlossinSiberianhamstersdespiteoverexpressionofthe agouti-relatedpeptide(AgRP)gene.JNeuroendocrinol2010;22:564–75. JethwaPH,WarnerA,NilaweeraKN,BrameldJM,KeyteJW,CarterWG,etal.

VGF-derivedpeptide,TLQP-21,regulatesfoodintakeandbodyweightinSiberian hamsters.Endocrinology2007;148:4044–55.

KayMA,GloriosoJC,NaldiniL.Viralvectorsforgenetherapy:theartofturning infectiousagentsintovehiclesoftherapeutics.NatMed2001;7:33–40. LewisJE,BrameldJM,JethwaPH.NeuroendocrineroleforVGF.FrontEndocrinol

2015:6.

MartinP,AlbagliO,PoggiMC,BoulukosKE,PognonecP.Developmentofanew bicistronicretroviralvectorwithstrongIRESactivity.BMCBiotechnol2006;6:4. NakaiH,StormTA,KayMA.IncreasingthesizeofrAAV-mediatedexpression cas-settesinvivobyintermolecularjoiningoftwocomplementaryvectors.Nat Biotechnol2000;18:527–32.

PahlmanS,RuusalaAI,AbrahamssonL,MattssonME,EsscherT.Retinoic acid-induceddifferentiationofculturedhumanneuroblastomacells:acomparison withphorbolester-induceddifferentiation.CellDiffer1984;14:135–44. PaxinosG,FranklinK.PaxinosandFranklin’sthemousebraininstereotaxic

coordi-nates.4thed.Amsterdam:Elsevier;2012.

PayneAJ,GerdesBC,KajaS,KoulenP.Insertsequencelengthdetermines transfec-tionefficiencyandgeneexpressionlevelsinbicistronicmammalianexpression vectors.IntJBiochemMolBiol2013;4:201–8.

RecchiaA,ParksRJ,LamartinaS,ToniattiC,PieroniL,PalomboF,etal.Site-specific integrationmediatedbyahybridadenovirus/adeno-associatedvirusvector. ProcNatlAcadSciUSA1999;96:2615–20.

RyanMD,DrewJ.Foot-and-mouthdiseasevirus2Aoligopeptidemediatedcleavage ofanartificialpolyprotein.EMBOJ1994;13:928–33.

RyanMD,KingAM,ThomasGP.Cleavageoffoot-and-mouthdiseasevirus polypro-teinismediatedbyresidueslocatedwithina19aminoacidsequence.JGenVirol 1991;72(Pt11):2727–32.

SweetmanD,GoljanekK,RathjenT,OustaninaS,BraunT,DalmayT,etal.Specific requirementsofMRFsfortheexpressionofmusclespecificmicroRNAs,miR-1, miR-206andmiR-133.DevBiol2008;321:491–9.

SzymczakAL,VignaliDA.Developmentof2Apeptide-basedstrategiesinthedesign ofmulticistronicvectors.ExpertOpinBiolTher2005;5:627–38.