The use of IRES-based bicistronic vectors allows the stable expression of recombinant G-protein coupled receptors such as NPY5 and histamine 4.
Stable expression of G protein coupled receptors in cell lines is a crucial tool for the characterization of the molecular pharmacology of receptors and the screening for new antagonists. However, in some instances, many difficulties have been encountered to obtain stable cell lines expressing functional receptors. Here, we addressed the question of vector optimization to establish cell lines expressing the human neuropeptide Y receptor 5 (NPY5-R) or histamine receptor 4 (HH4R). We have compared bicistronic vectors containing viral or cellular internal ribosome entry sites (IRES), co-expressing the receptor and the neomycine resistance gene from a single mRNA, to a bigenic vector containing two distinct promoters upstream each different genes. This study is the first one to validate the use of three cellular IRESs for long-term transgene expression. Our results demonstrate for both NPY5-R and HH4R that the bicistronic vectors with EMCV, VEGF, FGF1A or FGF2 IRES provide clones expressing functional receptors with yields between 25% and 100%. In contrast, the bigenic vector provided no functional clones, related to a low expression of NPY5R mRNA. The cell lines expressing active receptor were stable after more than 50 passages. These data indicate that IRES-based bicistronic vectors are particularly appropriate to establish cell clones expressing active G-coupled protein receptors with a high yield. In the case of NPY5, it was a new way to produce such a stable cell line. Furthermore, the characteristics-presented herein-of this receptor pharmacological property are perfectly in line with those reported in the literature.
Allera-Moreau
C
C
Institut National de la Santé et de la Recherche Médicale, INSERM, U589, Hormones, Facteurs de Croissance et Physiopathologie Vasculaire, Institut Louis-Bugnard, IFR31, Bâtiment L3, Avenue Jean-Poulhès, BP 84225, 31432 Toulouse cedex 04, France.
Chomarat
P
P
Audinot
V
V
Cogé
F
F
Gillard
M
M
Martineau
Y
Y
Boutin
J A
JA
Prats
A-C
AC
D015870:Gene Expression; D005796:Genes / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis* / Q000235:genetics*; D011968:Receptors, Histamine / Q000096:biosynthesis* / Q000235:genetics*; D000074040:Receptors, Histamine H4; D017476:Receptors, Neuropeptide Y / Q000096:biosynthesis* / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
C418607:HRH4 protein, human; D043562:Receptors, G-Protein-Coupled; D011968:Receptors, Histamine; D000074040:Receptors, Histamine H4; D017476:Receptors, Neuropeptide Y; D011994:Recombinant Proteins; C100568:neuropeptide Y5 receptor