Construction and isolation of recombinant vaccinia virus using genetic markers

The standard approach for the isolation of vaccinia virus recombinants involves homologous recombination between a transfected plasmid and the replicating viral DNA. In a typical infection/transfection experiment, recombinant viruses only account for a tiny proportion (10-4 to 10-3) of the progeny virus; thus, genetic markers are often included in the transfected plasmid to facilitate the selection of recombinant viruses. This chapter describes in detail two different selection procedures: one relies on plaque formation phenotype using the vaccinia virus gene F13L; the other relies on antibiotic resistance using the Escherichia coli xanthine-guanine phosphoribosyl transferase gene.