Gene expression using the vaccinia virus/ T7 RNA polymerase hybrid system.
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.
Elroy-Stein
O
O
Tel Aviv University, Tel Aviv, Israel.
Moss
B
B
D000818:Animals; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D014616:Vaccinia virus / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases