A simple method to generate stable cell lines for the analysis of transient protein-protein interactions
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 energ y transfer (BRET). This technique has been widely applied to study signaling pathways, in...
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2013-04-01
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| Series: | BioTechniques |
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| Online Access: | https://www.future-science.com/doi/10.2144/000114013 |
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| author | Emilia Elizabeth Savage Denise Wootten Arthur Christopoulos Patrick Michael Sexton Sebastian George Barton Furness |
| author_facet | Emilia Elizabeth Savage Denise Wootten Arthur Christopoulos Patrick Michael Sexton Sebastian George Barton Furness |
| author_sort | Emilia Elizabeth Savage |
| collection | DOAJ |
| description | 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 energ y 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. |
| format | Article |
| id | doaj-art-5bb5bd7b4e1948bbabdb49abc95b1af4 |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2013-04-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-5bb5bd7b4e1948bbabdb49abc95b1af42025-08-20T02:26:03ZengTaylor & Francis GroupBioTechniques0736-62051940-98182013-04-0154421722110.2144/000114013A simple method to generate stable cell lines for the analysis of transient protein-protein interactionsEmilia Elizabeth Savage0Denise Wootten1Arthur Christopoulos2Patrick Michael Sexton3Sebastian George Barton Furness41Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia1Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia1Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia1Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia1Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, AustraliaTransient 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 energ y 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.https://www.future-science.com/doi/10.2144/000114013BRETflpINGatewayprotein interactionbicistronic vectorarrestin |
| spellingShingle | Emilia Elizabeth Savage Denise Wootten Arthur Christopoulos Patrick Michael Sexton Sebastian George Barton Furness A simple method to generate stable cell lines for the analysis of transient protein-protein interactions BioTechniques BRET flpIN Gateway protein interaction bicistronic vector arrestin |
| title | A simple method to generate stable cell lines for the analysis of transient protein-protein interactions |
| title_full | A simple method to generate stable cell lines for the analysis of transient protein-protein interactions |
| title_fullStr | A simple method to generate stable cell lines for the analysis of transient protein-protein interactions |
| title_full_unstemmed | A simple method to generate stable cell lines for the analysis of transient protein-protein interactions |
| title_short | A simple method to generate stable cell lines for the analysis of transient protein-protein interactions |
| title_sort | simple method to generate stable cell lines for the analysis of transient protein protein interactions |
| topic | BRET flpIN Gateway protein interaction bicistronic vector arrestin |
| url | https://www.future-science.com/doi/10.2144/000114013 |
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