分子生物学英文文献1

75Chapter5AnEfficientProtocolforVZVBAC-BasedMutagenesisZhenZhang,YingHuang,andHuaZhuAbstractVaricella-zostervirus(VZV)causesbothvaricella(chickenpox)andherpeszoster(shingles).Asamemberofthehumanherpesvirusfamily,VZVcontainsalarge125-kbDNAgenome,encoding70uniqueopenreadingframes(ORFs).ThegeneticstudyofVZVhasbeenhinderedbythelargesizeofviralgenome,andthusthefunctionsofthemajorityoftheseORFsremainunclear.Recently,anefficientprotocolhasbeendevelopedbasedonaluciferase-containingVZVbacteriaartificialchromosome(BAC)systemtorapidlyisolateandstudyVZVORFdeletionmutants.Keywords:Varicella-zostervirus,Bacterialartificialchromosome,Deletionmutagenesis,BioluminescenceVaricella-zostervirus(VZV)isacommonhumanherpesvirusthatisasignificantpathogenintheUnitedStates,withmorethan90%oftheUSpopulationharboringthevirus(1).PrimaryinfectionofVZVleadstovaricella(chickenpox).VZVestablisheslifelonglatencyinthehost,specificallyintrigeminalgangliaanddorsalrootganglia(2).TheVZVreactivationresultsinherpeszoster(shingles),whichoftenleadstochronicpostherpeticneuralgia(2,3).Asamemberofhumanalpha-herpesvirussubfamily,VZVhasa125-kblongdouble-strandedDNAgenome,whichencodesatleast70uniqueopenreadingframes(ORFs).ThegenomesofseveraldifferentVZVstrainsweresequencedandafewoftheVZVgenesgeneticallyanalyzed(4).IthasbeenextremelydifficulttogenerateVZVsite-specificmutationsusingconventionalhomologyrecombinationmeth-ods.Thiswasmainlyduetothehighcell-associatednatureofVZVinfectioninvitro,whichleadstothedifficultiesinisolating1.IntroductionJeffBraman(ed.),InVitroMutagenesisProtocols:ThirdEdition,MethodsinMolecularBiology,vol.634,DOI10.1007/978-1-60761-652-8_5,©SpringerScience+BusinessMedia,LLC201076Zhang,Huang,andZhuviralDNAandpurifyingrecombinantvirusawayfromwild-typevirus.Inthelastfewyears,apopularmethodforVZVinvitromutagenesisinvolvesafour-cosmidsystemcoveringtheentireviralgenome(5–7).Usingthecosmidsystemtogeneraterecom-binantVZVvariantsinvolvestechnicallychallengingstepssuchasco-transfectionoffourlargecosmidsintopermissivemammaliancellsandmultiplehomologousrecombinationeventswithinasinglecelltoreconstructafull-lengthviralgenome.Thehighlycell-associatednatureofVZValsomakesthedownstreamappli-cationsoftraditionalvirologymethodssuchasplaqueassay-basedtiteringandplaquepurificationdifficult.Todate,thefunctionsofthemajorityofVZVORFsremainuncharacterized(8).InordertocreaterecombinantsofVZVmoreefficiently,thefull-lengthVZV(P-Okastrain,aclonedclinicalisolateofVZV)genomehasbeensuccessfullyclonedasaVZVbacteriaartificialchromosome(BAC)(9,10).ThisVZVBACcombinedwithahighlyefficientlyE.colihomologousrecombinationsystemallowsquickandeasygenerationofrecombinantVZV.Tofurthereasethedownstreamvirusquantificationassays,afireflyluciferasereportergene,wasinsertedintotheVZVBACtogenerateanovelluciferase-expressingVZV(10).Inthisprotocol,weshowthegenerationandanalysesofVZVfull-lengthORFdeletionmutantsandgeneticrevertantsasexamplestodemonstratetheutilityandefficiencyofthisversatilesystemforVZVmutagenesisinvitro.Furthermore,thisprotocolcanbeeasilymodifiedtobroadenitsapplicationstoavarietyofgeneticmaneuversincludingmakingdoubleORFdeletions,partialORFdeletions,insertions,andpointmutations.1.Humanmelanoma(MeWo)cellsweregrowninDMEMsupplementedwith10%fetalbovineserum,100Upenicillin–streptomycin/ml,and2.5mgamphotericinB/mlat37°Cinahumidifiedincubatorwith5%CO2.AlltissueculturereagentswereobtainedfromSigma(St.Louis,MO).2.VZVlucwasrecentlydevelopedinthelaboratory(10).Itcon-tainsafull-lengthVZVP-Okagenomewithafireflyluciferasecassette(seeNote1).TheBACvectorwasinsertedbetweenVZVORF60andORF61,whichincludesagreenfluorescentprotein(GFP)expressioncassetteandachloramphenicolresistancecassette(CmR).3.pGEM-oriV/kanwaspreviouslyconstructed(11)inthelab-oratoryandusedasaPCRtemplatetogeneratetheexpres-sioncassettesforthekanamycinorampicillinresistancegenes(KanRandAmpR).2.Materials2.1.Cells,VZVluc,Plasmids,andE.coliStrain77AnEfficientProtocolforVZVBAC-BasedMutagenesis4.pGEM-lox-zeowasderivedfrompGEM-T(Promega,Madison,WI)(12)andwasusedtogeneratetherescueclonesofVZVORFdeletionmutants.5.E.colistrainDY380wasobtainedfromNealCopelandandCraigStranthdeeandusedformutagenesis(13).6.AcrerecombinaseexpressionplasmidpGS403wasagiftfromL.Enquist(PrincetonUniversity,NJ).1.AllprimersweresynthesizedbySigma-Genosys(Woodlands,TX)andstoredinTEbuffer(100mM).2.HotStarTaqDNApolymerase(Qiagen,Valencia,CA)wasusedforPCRreactionsandPlatinumTaqDNApolymerase(Invitrogen,Carlsbad,CA)couldbeusedforoptionalhi-fidelityPCRreactions(seeNote2).3.PCRpurificationwascarriedoutusingaPCRpurificationkit(Qiagen,Valencia,CA).4.TheamplifiedlinearDNAsweresuspendedinsterileddH2Oandwerequantifiedbyspectroscopy(NanoDropTechnologies,Wilmington,DE).5.DpnI(NewEnglandBiolabs,Ipswich,MA)restrictiontreat-mentfollowingPCRwascarriedoutinordertoeliminatecirculartemplateDNA.6.ElectroporationwascarriedoutwithaGenePulserIIElectroporator(Bio-Rad,Hercules,CA).1.AllantibioticswereobtainedfromSigma(St.Louis,MO).LBplatescontainingspecificantibioticswereusedforappro-priates