Eukaryotic expression vectors resistant to transgene silencing

Abstract

The present invention relates to recombinant DNA molecules such as plasmids, non-viral vectors, viral vectors and hybrids thereof, and more particularly to vector modifications that improve expression of said DNA molecules in cell lines and organisms.

1 - 13 . (canceled) 
     
     
         14 . An expression vector comprising a prokaryotic replication origin, a selectable marker, a eukaryotic expression cassette and tRNA promoters upstream and downstream of said eukaryotic expression cassette, said tRNA promoters each comprising the two intragenic promoter elements RNA pol III type II promoter A-box and B-box, said eukaryotic expression cassette comprising a transgene under the control of a heterologous promoter. 
     
     
         15 . The expression vector of  claim 14 , wherein said selectable marker comprises an antibiotic free selectable marker. 
     
     
         16 . The expression vector of  claim 14 , wherein said vector is selected from the group consisting of non-viral and viral vectors. 
     
     
         17 . The expression vector of  claim 14 , wherein said vector is a plasmid. 
     
     
         18 . Vertebrate cells transfected with an expression vector, said expression vector comprising a prokaryotic replication origin, a selectable marker, a eukaryotic expression cassette and tRNA promoters upstream and downstream of said eukaryotic expression cassette, said tRNA promoters each comprising the two intragenic promoter elements RNA pol III type II promoter A-box and B-box, said eukaryotic expression cassette comprising a transgene under the control of a heterologous promoter. 
     
     
         19 . The vertebrate cells of  claim 18 , wherein said tRNA promoters prevent gene silencing of said transgene. 
     
     
         20 . The vertebrate cells of  claim 18 , wherein said selectable marker comprises an antibiotic free selectable marker. 
     
     
         21 . The vertebrate cells of  claim 18 , wherein said vector is selected from the group consisting of non-viral and viral vectors. 
     
     
         22 . The vertebrate cells of  claim 21 , wherein said vector is a plasmid. 
     
     
         23 . The vertebrate cells of  claim 18 , wherein said vector is selected from the group consisting of non-integrating vectors and integrating vectors.