title: Engineering recombinant poxviruses using a compact GFP-blasticidin resistance fusion gene for selection.
abstract: Recombinant poxviruses are important tools for research and some are candidate vaccines. To make these viruses a simple, small vector that can be used to engineer multiple strains of vaccinia virus and other model poxviruses, including ectromelia virus is of value. Here a set of plasmids and methods for making these viruses that uses an enhanced green fluorescent protein-blasticidin resistance (GFP-bsd) fusion gene as a transient selectable marker are described. This gene is smaller than any of the bi-functional selection markers used previously. The versatility of the method across different poxviruses is demonstrated by engineering changes into multiple loci of the WR and Modified Vaccinia Ankara (MVA) strains of vaccinia virus and also ectromelia virus. Finally, a set of vaccinia virus sequences for directing homologous recombination that are very highly conserved was designed and tested. These sequences allow a single plasmid to be used to insert a transgene into multiple strains of the virus.
authors.affiliation: Research School of Biology, The Australian National University, Canberra, ACT, Australia.
authors.forename: Yik Chun
authors.initials: YC
authors.lastname: Wong
authors.forename: Leon C W
authors.initials: LC
authors.lastname: Lin
authors.forename: Carolina R
authors.initials: CR
authors.lastname: Melo-Silva
authors.forename: Stewart A
authors.initials: SA
authors.lastname: Smith
authors.forename: David C
authors.initials: DC
authors.lastname: Tscharke
mesh_terms: D000619:Aminohydrolases / Q000235:genetics; D000998:Antiviral Agents / Q000494:pharmacology; D024882:Drug Resistance, Viral; D004481:Ectromelia virus / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D009705:Nucleosides / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012641:Selection, Genetic; D013194:Staining and Labeling / Q000379:methods*; D014616:Vaccinia virus / Q000235:genetics*; D014773:Virology / Q000379:methods*
chemical_list: D000998:Antiviral Agents; D009705:Nucleosides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C004500:blasticidin S; D000619:Aminohydrolases; C019795:blasticidin S deaminase