Comparative analysis of internal ribosomal entry sites as molecular tools for bicistronic expression

• Subtle alterations of the EMCV IRES can drastically reduce second cistron expression.
• The EMCV IRES can mediate much higher expression than the VCIP and Gtx IRESes.
• A cloning-friendly variation of the EMCV IRES performs comparably with the wild type element.

Internal ribosomal entry sites (IRESes) are sequences that drive cap-independent translation. They are found in some viral and cellular transcripts and they have been extensively used in both basic and applied research for the translation of two or more polypeptides from a single mRNA molecule in eukaryotic cells. Although the most widely used IRES comes from the encephalomyocarditis virus (EMCV), several other viral and cellular IRES elements have been identified and successfully used, including those of the human VCIP gene and the mouse Gtx gene. In this report we have compared the EMCV IRES with the VCIP and the Gtx IRESes, and we provide evidence that by using the EMCV IRES much higher levels of second cistron expression can be achieved.