title: Alternative-splicing-based bicistronic vectors for ratio-controlled protein expression and application to recombinant antibody production.
abstract: 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.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale, U858, CHU Rangueil, BP 84225, 31432 Toulouse cedex 4, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Fallot
authors.forename: Raouia
authors.initials: R
authors.lastname: Ben Naya
authors.forename: Corinne
authors.initials: C
authors.lastname: Hieblot
authors.forename: Philippe
authors.initials: P
authors.lastname: Mondon
authors.forename: Eric
authors.initials: E
authors.lastname: Lacazette
authors.forename: Khalil
authors.initials: K
authors.lastname: Bouayadi
authors.forename: Abdelhakim
authors.initials: A
authors.lastname: Kharrat
authors.forename: Christian
authors.initials: C
authors.lastname: Touriol
authors.forename: Hervé
authors.initials: H
authors.lastname: Prats
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics*; D006224:Cricetinae; D005822:Genetic Vectors; D006801:Humans; D008156:Luciferases / Q000032:analysis; D011132:Polyribosomes / Q000378:metabolism; D022821:RNA Splice Sites; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D022821:RNA Splice Sites; D012333:RNA, Messenger; D011994:Recombinant Proteins; D008156:Luciferases