Formation of AAV Single Stranded DNA Genome from a Circular Plasmid in Saccharomyces cerevisiae 英文参考文献.docVIP

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Formation of AAV Single Stranded DNA Genome from a Circular Plasmid in Saccharomyces cerevisiae 英文参考文献.doc

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Formation of AAV Single Stranded DNA Genome from a Circular Plasmid in Saccharomyces cerevisiae 英文参考文献

FormationofAAVSingleStrandedDNAGenomefroma CircularPlasmidinSaccharomycescerevisiae TizianaCervelli1,AnaBackovic2,AlvaroGalli1* 1LaboratoriodiTerapiaGenicaeMolecolare,IstitutodiFisiologiaClinica,CNR,Pisa,Italy,2LaboratoriodiBiologiaMolecolare,ScuolaNormaleSuperiore,Pisa,Italy Abstract Adeno-associatedvirus(AAV)-basedvectorsarepromisingtoolsfortargetedtransferingenetherapystudies.Manyefforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly,forAAVvectorproduction.IthasbeenshownthatyeastSaccharomycescerevisiaeisabletoreplicateandform thecapsidofmanyviruses.WeinvestigatedtheabilityoftheyeastSaccharomycescerevisiaetocarryoutthereplicationofa recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiaeURA3gene,wasco-transformedinyeastwithaplasmidexpressingRep68,asignificantnumberofURA3+clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealedthatsinglestrandedDNAisformedandthattheplasmidisentirelyreplicated.ThessDNAcontainstheITRs,URA3 geneandalsovectorsequencessuggestingthepresenceoftwodistinctmolecules.ItsformationwasdependentonRep68 expressionandITR.ThesedataindicatethatDNAisnotobtainedbythecanonicalAAVreplicationpathway. Citation:CervelliT,BackovicA,GalliA(2011)FormationofAAVSingleStrandedDNAGenomefromaCircularPlasmidinSaccharomycescerevisiae.PLoSONE6(8): e23474.doi:10.1371/journal.pone.0023474 Editor:ArthurJ.Lustig,TulaneUniversityHealthSciencesCenter,UnitedStatesofAmerica ReceivedMarch7,2011;AcceptedJuly19,2011;PublishedAugust10,2011 Copyright: ? 2011 Cervelli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse,distribution,andreproductioninanymedium,providedt