Transfer of the inducible lac repressor/operator system from Escherichia coli to a vaccinia virus expression vector.

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Abstract
Cis- and trans-acting elements of the Escherichia coli lac operon were transferred to vaccinia virus and used to regulate gene expression. A recombinant virus that constitutively expresses a modified lac repressor gene (lacI) was constructed. We calculated that each infected cell contained .apprxeq. 2 .times. 107 active repressor molecules (and 1-2 .times. 104 copies of the vaccinia virus genome). A strong vaccinia-virus late promoter was modified by insertion of the lac operator (lacO) at various positions. The ability of each modified promoter to regulate expression of .beta.-galactosidase was tested by transient assays in cells infected with wild-type or lacI-containing vaccinia virus. Placement of the lacO just downstream of the conserved TAAAT sequence of a late promoter was consistent with a minimal effect on basal expression and good repressibility, whereas basal expression was severely inhibited when lacO overlapped or preceded the TAAAT motif. A single recombinant vaccinia virus containing lacI and the .beta.-galactosidase gene under control of the optimal lacO promoter was contructed. In the absence of inducer, cells infected with this double recombinant virus synthesized little or no detectable .beta.-galactosidase. Addition of isopropyl .beta.-D-thiogalactoside restored expression to > 20% of the unrepressed level. This inducible vector system has potential applications for expression of heterologous and homologous genes.