Vaccinia virus vectors utilizing the beta-galactosidase assay for rapid selection of recombinant viruses and measurement of gene expression

Abstract

Plasmids were constructed fusing vaccinia transcriptional regulatory sequences (promoters) to the lacZ gene of Escherichia coli. These recombinant plasmids were used to compare relative promoter strengths in transient expression assays and to construct recombinant vaccinia viruses producing beta-galactosidase (beta Gal). Viruses synthesizing beta Gal were determined by utilizing the chromogenic substrate, 5-bromo-4-chloro-3-indoyl-beta-D-galactoside to form blue plaques. A recombinant virus producing beta Gal was then used to select a second recombinant virus. This was accomplished via in vivo recombination replacing the lacZ gene with a sequence coding for the gp85 protein of Friend murine leukemia virus. The recombinant virus was selected by its inability to form blue plaques under appropriate conditions.