Vaccinia virus expression vector: coexpression of beta-galactosidase provides visual screening of recombinant virus plaques.
We constructed a plasmid coexpression vector that directs the insertion of a foreign gene of interest together with the Escherichia coli beta-galactosidase (beta gal) gene into the thymidine kinase (TK) locus of the vaccinia virus genome. Tissue culture cells that had been infected with vaccinia virus were transfected with a plasmid vector containing a foreign gene. TK- recombinants could be selected by a plaque assay on TK- cells in the presence of 5-bromodeoxyuridine and distinguished from spontaneous TK- mutants by the addition of a beta-gal indicator to the agarose overlay. Plaques that expressed beta-gal stained dark blue within several hours at 37 degrees C. Alternatively, TK- selection could be eliminated, and recombinant plaques could be readily identified solely by their blue color. The reverse procedure, in which the starting virus expresses beta-gal (i.e., forms blue plaques) and the desired recombinant has deleted the entire beta-gal gene (i.e., forms white plaques), is another alternative. Each protocol was tested by constructing vaccinia virus recombinants that express hepatitis B virus surface antigen.
D004926:Escherichia coli / Q000235:genetics; D005696:Galactosidases / Q000235:genetics*; D005822:Genetic Vectors; D006514:Hepatitis B Surface Antigens / Q000235:genetics; D009154:Mutation; D010858:Pigmentation; D010957:Plasmids; D011995:Recombination, Genetic; D013937:Thymidine Kinase / Q000235:genetics; D014616:Vaccinia virus / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics*
D006514:Hepatitis B Surface Antigens; D013937:Thymidine Kinase; D005696:Galactosidases; D001616:beta-Galactosidase