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US 5,135,855 AGranted
Rapid, versatile and simple system for expressing genes in eukaryotic cells
AssigneeThe United States of America as represented by the Department of Health and Human Services
InventorsBernard Moss, Thomas Fuerst, Orna Elroy-Stein
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Abstract
An efficient, versatile and simple expression system which confers cap-independent translation to prokaryotic RNAs in eukaryotic cells has been described. The utility of recombinant vaccinia virus for this purpose has been illustrated.
Claims12
A method for synthesizing protein, comprising:
- (a) infecting a mammalian cell with a recombinant cytoplamic DNA virus the genome of which includes the following elements operably linked 5' to 3':
- a promoter, wherein said promoter is selected from the group consisting of T7, SP6, T3, and GH1 promoters, followed by a cDNA copy of an untranslated region of a cardiovirus RNA that confers cap-independent translation, followed by a gene encoding a protein desired to be expressed, said promoter, cDNA and the gene being so arranged that said gene is efficiently expressed in a mammalian cell infected with said recombinant virus only when said cell contains an RNA polymerase active with said promoter;
- and
- (b) infecting said cell with a second cytoplasmic DNA virus that expresses said RNA polymerase so that said protein is expressed in the cell;
- and then
- (c) recovering the desired protein by suitable isolation and purification techniques.
3 more elements
A method for synthesizing protein, which comprises transfecting a mammalian cell with a recombinant plasmid comprising the following elements operably linked 5' to 3;:
- a promoter, wherein said promoter is selected from the group consisting of T7, SP6, T3, and GH1 promoters, followed by a cDNA copy of an untranslated region of a cardiovirus RNA that confers cap-independent translation, followed by a gene encoding a protein desired to be expressed, said promoter, cDNA and the gene being so arranged that said gene is efficiently expressed in a mammalian cell transfected with said recombinant plasmid only when said cell contains RNA polymerase active with said promoter;
- and infecting said cell with a DNA based cytoplasmic virus expressing said RNA polymerase so that said protein is expressed in said cell;
- and then recovering the desired protein by suitable isolation and purification techniques.
Dependencies are inferred from the claim text.