Partial and Full PCR-Based Reverse Genetics Strategy for Influenza Viruses 英文参考文献.docVIP

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Partial and Full PCR-Based Reverse Genetics Strategy for Influenza Viruses 英文参考文献.doc

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Partial and Full PCR-Based Reverse Genetics Strategy for Influenza Viruses 英文参考文献

PartialandFullPCR-BasedReverseGeneticsStrategyfor InfluenzaViruses HongjunChen,JianqiangYe,KeminXu,MatthewAngel,HongxiaShao,AndreaFerrero,TroySutton, DanielR.Perez* Virginia-MarylandRegionalCollegeofVeterinaryMedicine,DepartmentofVeterinaryMedicine,UniversityofMaryland,CollegePark,Maryland,UnitedStatesofAmerica Abstract Since 1999, plasmid-based reverse genetics (RG) systems have revolutionized the way influenza viruses are studied. However,itisnotunusualtoencountercloningdifficultiesforoneormoreinfluenzageneswhileattemptingtorecover virusdenovo.Toovercomesomeoftheseshortcomingswesoughttodeveloppartialorfullplasmid-freeRGsystems.The influenzageneofchoiceisassembledintoaRGcompetentunitbyvirtueofoverlappingPCRreactionscontainingacDNA copyoftheviralgenesegmentunderthecontrolofRNApolymeraseIpromoter(pol1)andtermination(t1)signals–herein referredtoasFluPCRamplicons.TransfectionoftissueculturecellswitheitherHAorNAFluPCRampliconsand7plasmids encodingtheremaininginfluenzaRGunits,resultedinefficientvirusrescue.Likewise,transfectionsincludingbothHAand NAFluPCRampliconsand6RGplasmidsalsoresultedinefficientvirusrescue.Inaddition,influenzaviruseswererecovered fromafullsetofFluPCRampliconswithouttheuseofplasmids. Citation:ChenH,YeJ,XuK,AngelM,ShaoH,etal.(2012)PartialandFullPCR-BasedReverseGeneticsStrategyforInfluenzaViruses.PLoSONE7(9):e46378. doi:10.1371/journal.pone.0046378 Editor:EarlG.Brown,UniversityofOttawa,Canada ReceivedJuly23,2012;AcceptedAugust29,2012;PublishedSeptember28,2012 Copyright: ? 2012 Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding: Theresearchwas funded bycontract Nu HHSN266200700010C fromtheNationalInstitute ofAllergyandInfectiousDiseases, NationalInstitutesof Health.Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationoftheman