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.
|Journal||Current Protocols in Molecular Biology|
|State||Published - May 2001|