title: A simple method to generate stable cell lines for the analysis of transient protein-protein interactions.
abstract: Transient protein-protein interactions form the basis of signal transduction pathways in addition to many other biological processes. One tool for studying these interactions is bioluminescence resonance energy transfer (BRET). This technique has been widely applied to study signaling pathways, in particular those initiated by G protein-coupled receptors (GPCRs). These assays are routinely performed using transient transfection, a technique that has limitations in terms of assay cost and variability, overexpression of interacting proteins, vector uptake limited to cycling cells, and non-homogenous expression across cells within the assay. To address these issues, we developed bicistronic vectors for use with Life Technology's Gateway and flpIN systems. These vectors provide a means to generate isogenic cell lines for comparison of interacting proteins. They have the advantage of stable, single copy, isogenic, homogeneous expression with low inter-assay variation. We demonstrate their utility by assessing ligand-induced interactions between GPCRs and arrestin proteins.
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.
authors.forename: Emilia Elizabeth
authors.initials: EE
authors.lastname: Savage
authors.forename: Denise
authors.initials: D
authors.lastname: Wootten
authors.forename: Arthur
authors.initials: A
authors.lastname: Christopoulos
authors.forename: Patrick Michael
authors.initials: PM
authors.lastname: Sexton
authors.forename: Sebastian George Barton
authors.initials: SG
authors.lastname: Furness
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008024:Ligands; D008163:Luminescent Measurements / Q000379:methods; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D025941:Protein Interaction Mapping / Q000379:methods*; D043562:Receptors, G-Protein-Coupled / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods*
chemical_list: D019390:Arrestins; D008024:Ligands; D008164:Luminescent Proteins; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins