A Selectable and Excisable Marker System for the Rapid Creation of Recombinant Poxviruses 英文参考文献.docVIP

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A Selectable and Excisable Marker System for the Rapid Creation of Recombinant Poxviruses 英文参考文献.doc

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A Selectable and Excisable Marker System for the Rapid Creation of Recombinant Poxviruses 英文参考文献

ASelectableandExcisableMarkerSystemfortheRapid CreationofRecombinantPoxviruses JuliaL.Rintoul1,2.,JiahuWang2.,DonB.Gammon3,NicholasJ.vanBuuren3,KennethGarson2 ,Karen Jardine2,MicheleBarry3,DavidH.Evans3,JohnC.Bell1,2* 1Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Canada, 2Centre for Cancer Therapeutics, Ottawa HospitalResearchInstitute,Ottawa,Canada,3DepartmentofMedicalMicrobiologyandImmunology,FacultyofMedicineandDentistry,LiKaShingInstituteofVirology, UniversityofAlberta,Edmonton,Canada Abstract Background:Geneticmanipulationofpoxvirusgenomesthroughattenuation,orinsertionoftherapeuticgeneshasledtoa numberofvectorcandidatesforthetreatmentofavarietyofhumandiseases.Thedevelopmentofrecombinantpoxviruses ofteninvolvesthegenomicinsertionofaselectablemarkerforpurificationandselectionpurposes.Theuseofmarkergenes howeverinevitablyresultsinavectorthatcontainsunwantedgeneticinformationofnotherapeuticvalue. Methodology/Principal Findings: Here we describe an improved strategy that allows for the creation of marker-free recombinant poxviruses of any species. The Selectable and Excisable Marker (SEM) system incorporates a unique fusion marker gene for the efficient selection of poxvirus recombinants and the Cre/loxP system to facilitate the subsequent removalofthemarker.Wehavedefinedandcharacterizedthisnewmethodologicaltoolbyinsertionofaforeigngeneinto vacciniavirus,withthesubsequentremovaloftheselectablemarker.WethenanalyzedtheimportanceofloxPorientation duringCrerecombination,andshowthattheSEMsystemcanbeusedtointroducesite-specificdeletionsorinversionsinto theviralgenome.Finally,wedemonstratethattheSEMstrategyisamenabletootherpoxviruses,asdemonstratedhere withthecreationofanectromeliavirusrecombinantlackingtheEVM002gene. Conclusion/Significance: The system described here thus provides a faster, simpler and more efficient means to create clinic-readyrecombinantpoxvirusesfortherapeuticgenetherapyapplications. Citati