Efficient targeted insertion of an unselected marker into the vaccinia virus genome

A method is described by which an unselected marker can be inserted into the vaccinia virus genome. Cells were infected with defective virus (either isatin-beta-thiosemicarbazone dependent or temperature sensitive) and then cotransformed with a mixture of full-length wild-type genomic vaccinia virus DNA and a cloned vaccinia DNA molecule containing an allele for phosphonoacetic acid resistance. After incubation under conditions which are nonpermissive for the defective virus but which do not select for incorporation of phosphonoacetic acid resistance, the virus yields were assayed for the presence of all markers involved. Phosphonoacetic acid resistance was inserted into an otherwise wild-type genome with an efficiency of 25-33%. This represents an increase in efficiency of 150-to 3000-fold relative to controls. The procedure should be extremely useful for engineering the vaccinia virus genome.