Gene Expression Using the Vaccinia Virus/T7 RNA Polymerase Hybrid System

Abstract This unit describes a transient cytoplasmic expression system that relies on the synthesis of the bacteriophage T7 RNA polymerase in the cytoplasm of mammalian cells. A gene of interest is inserted into a plasmid such that it comes under the control of the T7 RNA polymerase promoter ( p T7 ). Using liposome‐mediated transfection, this recombinant plasmid is introduced into the cytoplasm of cells infected with vTF7‐3, a recombinant vaccinia virus encoding bacteriophage T7 RNA polymerase. During incubation, the gene of interest is transcribed with high efficiency by T7 RNA polymerase. For large‐scale work, protocols are provided for insertion of the p T7 ‐regulated gene into a second recombinant vaccinia virus by homologous recombination and subsequent coinfection with vTF7‐3 into cells grown in suspension or for direct transfection into OST7‐1 cells (a stable cell line that constitutively expresses the T7 RNA polymerase). Expressed protein is then analyzed by pulse‐labeling and purified. One new development to this vaccinia virus/T7 RNA polymerase hybrid expression system described here is the VOTE inducible expression system, which eliminates the need to use two recombinant viruses or a special cell line.