A simple method to generate stable cell lines for the analysis of transient protein-protein interactions

Research output: Contribution to journalArticleResearchpeer-review

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 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.
Original languageEnglish
Pages (from-to)217 - 221
Number of pages5
JournalBioTechniques
Volume54
Issue number4
DOIs
Publication statusPublished - 2013

Cite this

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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 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.",
author = "Savage, {Emilia Elizabeth} and Wootten, {Denise Laura} and Arthur Christopoulos and Patrick Sexton and Furness, {Sebastian George Barton}",
year = "2013",
doi = "10.2144/000114013",
language = "English",
volume = "54",
pages = "217 -- 221",
journal = "BioTechniques",
issn = "0736-6205",
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}

A simple method to generate stable cell lines for the analysis of transient protein-protein interactions. / Savage, Emilia Elizabeth; Wootten, Denise Laura; Christopoulos, Arthur; Sexton, Patrick; Furness, Sebastian George Barton.

In: BioTechniques, Vol. 54, No. 4, 2013, p. 217 - 221.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - A simple method to generate stable cell lines for the analysis of transient protein-protein interactions

AU - Savage, Emilia Elizabeth

AU - Wootten, Denise Laura

AU - Christopoulos, Arthur

AU - Sexton, Patrick

AU - Furness, Sebastian George Barton

PY - 2013

Y1 - 2013

N2 - 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.

AB - 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.

UR - http://www.ncbi.nlm.nih.gov/pubmed/23581469

U2 - 10.2144/000114013

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