Reverse guanine phosphoribosyltransferase selection of recombinant vaccinia viruses

We have developed a procedure for the selection of recombinant vaccinia viruses with applicability to poxvirus mutagenesis studies and to the use of vaccinia virus as an expression vector. The method depends on the specific inability of a recombinant vaccinia virus expressing the Escherichia coli guanine phosphoriboxyltransferase gene (gpt) to form plaques on a hypoxanthine-guanine phosphoribosyltransferase-negative line of mouse fibroblasts in the presence of 6-thioguanine. Recombinant viruses that have the gpt removed can form plaques under selection conditions, thus providing a simple and efficient selection protocol. We have demonstrated the method by isolating a pseudo-wild type revertant virus and a simple deletion mutant virus from a recombinant vaccinia virus with gpt inserted into the vaccinia virus gene encoding the major 35,000-Da secretory protein.