Efficient bicistronic expression of cre in mammalian cells

Cre recombinase-mediated DNA recombination is proving to be a powerful tech nique for the generation of mosaic mutant mice. To develop this technology further, we have altered the cre gene to enhance its expression in mammalia n cells and have tested its efficiency of expression in a bicistronic messa ge. Using a transient transfection assay, we found that the extension of a eukaryotic translation initiation consensus sequence, the insertion of two N-terminal amino acids, and the mutation of a cryptic splice acceptor site did not detectably alter Cre recombinase activity. The addition of either o f two introns resulted in an similar to 2-fold increase in recombination fr equency. We then tested the relative efficacy of Cre-mediated recombination in several bicistronic messages having the encephalomyocarditis virus inte rnal ribosome entry site (IRES), Recombination frequencies were only reduce d 2-fold relative to a comparable monocistronic cre gene. The latter result s indicate that it will be possible to generate transgenic mouse strains ha ving tissue-specific expression of the Cre recombinase through integration of an IRES-cre gene without disabling the targeted gene.