Dominant selection of vaccinia recombinants by co-transfection with a neomycin resistance gene

Abstract

Summary A recombinant vaccinia virus containing the neomycin resistance gene of the bacterial transposon Tn5 with an upstream vaccinia promoter inserted into the vaccinia thymidine kinase (TK) gene was constructed. Its genomic DNA was isolated and characterized. Expression of the aminoglycoside-3′-phosphotransferase, demonstrated by an enzymatic assay, was higher than that in cells transformed by the neomycin resistance gene. It was shown that the recombinant was about ten times more resistant than the wild type virus in plaque formation in 1D cells treated with G418. The use of G418 resistance as a dominant marker for co-transfection with another foreign gene was then directly demonstrated. This was done by constructing appropriate plasmids containing the neomycin resistance gene and also the herpes simplex virus TK gene with another vaccinia promoter placed upstream. These plasmids were used to construct two different vaccinia recombinants by transfection of cells which had been infected with corresponding vaccinia viruses. Recombinants, which expressed both genes, were selected simply by serial passages in G418-containing medium. Thus, co-transfection of a foreign gene with the neomycin resistance gene facilitates the use of vaccinia virus as a vector by providing a novel selection tool.