Nucleic Acids Research Advance Access originally published online on September 3, 2009
Nucleic Acids Research 2009 37(20):e134; doi:10.1093/nar/gkp716
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Nucleic Acids Research, 2009, Vol. 37, No. 20 e134
© The Author(s) 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
Alternative-splicing-based bicistronic vectors for ratio-controlled protein expression and application to recombinant antibody production
1Institut National de la Santé et de la Recherche Médicale (INSERM), U858, CHU Rangueil, BP 84225, 31432 Toulouse cedex 4, 2Millegen, Immeuble BIOSTEP, Bâtiment A, Rue Pierre et Marie Curie, BP38183, 31681 Labège cedex and 3Université Toulouse III Paul-Sabatier, Institut de Médecine Moléculaire de Rangueil, Equipe n°15, IFR31, Toulouse, France
*To whom correspondence should be addressed. Tel: +33 561 32 21 46; Fax: +33 561 32 21 41; Email: herve.prats{at}inserm.fr
Received May 25, 2009. Revised July 29, 2009. Accepted August 14, 2009.
In the last decade polycistronic vectors have become essential tools for both basic science and gene therapy applications. In order to co-express heterologous polypeptides, different systems have been developed from Internal Ribosome Entry Site (IRES) based vectors to the use of the 2A peptide. Unfortunately, these methods are not fully suitable for the efficient and reproducible modulation of the ratio between the proteins of interest. Here we describe a novel bicistronic vector type based on the use of alternative splicing. By modifying the consensus sequence that governs splicing, we demonstrate that the ratio between the synthesized proteins could easily vary from 1 : 10 to 10 : 1. We have established this system with luciferase genes and we extended its application to the production of recombinant monoclonal antibodies. We have shown that these vectors could be used in several typical cell lines with similar efficiencies. We also present an adaptation of these vectors to hybrid alternative splicing/IRES constructs that allow a ratio-controlled expression of proteins of interest in stably transfected cell lines.