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Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying
Sensitive simultaneous assessment of multiple signaling pathways within the same cells requires orthogonal reporters that can assay over large dynamic ranges. Luciferases are such genetically encoded candidates due to their sensitivity, versatility, and cost-effectiveness. We expand luciferase multi...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6911020/ https://www.ncbi.nlm.nih.gov/pubmed/31836712 http://dx.doi.org/10.1038/s41467-019-13651-y |
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author | Sarrion-Perdigones, Alejandro Chang, Lyra Gonzalez, Yezabel Gallego-Flores, Tatiana Young, Damian W. Venken, Koen J. T. |
author_facet | Sarrion-Perdigones, Alejandro Chang, Lyra Gonzalez, Yezabel Gallego-Flores, Tatiana Young, Damian W. Venken, Koen J. T. |
author_sort | Sarrion-Perdigones, Alejandro |
collection | PubMed |
description | Sensitive simultaneous assessment of multiple signaling pathways within the same cells requires orthogonal reporters that can assay over large dynamic ranges. Luciferases are such genetically encoded candidates due to their sensitivity, versatility, and cost-effectiveness. We expand luciferase multiplexing in post-lysis endpoint luciferase assays from two to six. Light emissions are distinguished by a combination of distinct substrates and emission spectra deconvolution. All six luciferase reporter units are stitched together into one plasmid facilitating delivery of all reporter units through a process we termed solotransfection, minimizing experimental errors. We engineer a multiplex hextuple luciferase assay to probe pathway fluxes through five transcriptional response elements against a control constitutive promoter. We can monitor effects of siRNA, ligand, and chemical compound treatments on their target pathways along with the four other probed cellular pathways. We demonstrate the effectiveness and adaptiveness of multiplex luciferase assaying, and its broad application across different research fields. |
format | Online Article Text |
id | pubmed-6911020 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-69110202019-12-16 Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying Sarrion-Perdigones, Alejandro Chang, Lyra Gonzalez, Yezabel Gallego-Flores, Tatiana Young, Damian W. Venken, Koen J. T. Nat Commun Article Sensitive simultaneous assessment of multiple signaling pathways within the same cells requires orthogonal reporters that can assay over large dynamic ranges. Luciferases are such genetically encoded candidates due to their sensitivity, versatility, and cost-effectiveness. We expand luciferase multiplexing in post-lysis endpoint luciferase assays from two to six. Light emissions are distinguished by a combination of distinct substrates and emission spectra deconvolution. All six luciferase reporter units are stitched together into one plasmid facilitating delivery of all reporter units through a process we termed solotransfection, minimizing experimental errors. We engineer a multiplex hextuple luciferase assay to probe pathway fluxes through five transcriptional response elements against a control constitutive promoter. We can monitor effects of siRNA, ligand, and chemical compound treatments on their target pathways along with the four other probed cellular pathways. We demonstrate the effectiveness and adaptiveness of multiplex luciferase assaying, and its broad application across different research fields. Nature Publishing Group UK 2019-12-13 /pmc/articles/PMC6911020/ /pubmed/31836712 http://dx.doi.org/10.1038/s41467-019-13651-y Text en © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Sarrion-Perdigones, Alejandro Chang, Lyra Gonzalez, Yezabel Gallego-Flores, Tatiana Young, Damian W. Venken, Koen J. T. Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying |
title | Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying |
title_full | Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying |
title_fullStr | Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying |
title_full_unstemmed | Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying |
title_short | Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying |
title_sort | examining multiple cellular pathways at once using multiplex hextuple luciferase assaying |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6911020/ https://www.ncbi.nlm.nih.gov/pubmed/31836712 http://dx.doi.org/10.1038/s41467-019-13651-y |
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