title: Improved vectors for stable expression of foreign genes in mammalian cells by use of the untranslated leader sequence from EMC virus.
abstract: Dicistronic mRNA expression vectors efficiently translate a 5' open reading frame (ORF) and contain a selectable marker within the 3' end which is inefficiently translated. In these vectors, the efficiency of translation of the selectable 3' ORF is reduced approximately 100-fold and is highly dependent on the particular sequences inserted into the 5' cloning site. Upon selection for expression of the selection marker gene product, deletions within the 5' ORF occur to yield more efficient translation of the selectable marker. We have generated improved dicistronic mRNA expression vectors by utilization of a putative internal ribosomal entry site isolated from encephalomyocarditis (EMC) virus. Insertion of the EMC virus leader sequence upstream of an ORF encoding either a wildtype or methotrexate resistant dihydrofolate reductase (DHFR) reduces DHFR translation up to 10-fold in a monocistronic DHFR expression vector. However, insertion of another ORF upstream of the EMC leader to produce a dicistronic mRNA does not further reduce DHFR translation. In the presence of the EMC virus leader, DHFR translation is not dependent on sequences inserted into the 5' end of the mRNA. We demonstrate that stable high level expression of inserted cDNAs may be rapidly achieved by selection for methotrexate resistance in DHFR deficient as well as DHFR containing cells. In contrast to previously described dicistronic expression vectors, these new vectors do not undergo rearrangement or deletion upon selection for amplification by propagation in increasing concentrations of methotrexate. The explanation may be either that the EMC virus leader sequence allows internal initiation of translation or that cryptic splice sites in the EMC virus sequence mediate production of monocistronic mRNAs. These vectors may be generally useful to rapidly obtain high level expression of cDNA genes in mammalian cells.
authors.affiliation: Genetics Institute, Cambridge, MA 02140.
authors.forename: R J
authors.initials: RJ
authors.lastname: Kaufman
authors.forename: M V
authors.initials: MV
authors.lastname: Davies
authors.forename: L C
authors.initials: LC
authors.lastname: Wasley
authors.forename: D
authors.initials: D
authors.lastname: Michnick
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004686:Encephalomyocarditis virus / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D000882:Haplorhini; D019868:Kanamycin Kinase; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010770:Phosphotransferases / Q000235:genetics; D014176:Protein Biosynthesis; D051381:Rats; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014162:Transfection
chemical_list: D013762:Tetrahydrofolate Dehydrogenase; D010770:Phosphotransferases; D019868:Kanamycin Kinase