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.
Isaacs
S N
SN
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Kotwal
G J
GJ
Moss
B
B
D000818:Animals; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D051379:Mice; D009154:Mutation; D011212:Poxviridae / Q000235:genetics; D011995:Recombination, Genetic; D014407:Tumor Cells, Cultured; D014616:Vaccinia virus / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D010948:Viral Plaque Assay
D007041:Hypoxanthine Phosphoribosyltransferase