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Reporter gene comparison demonstrates interference of complex body fluids with secreted luciferase activity
Reporter gene assays are widely used to study cellular signaling and transcriptional activity. Few studies describe the use of reporter genes for studying cellular responses on complex body fluids, such as urine and blood. Selection of the optimal reporter gene is crucial for study outcome. Here, we...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7809208/ https://www.ncbi.nlm.nih.gov/pubmed/33446782 http://dx.doi.org/10.1038/s41598-020-80451-6 |
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author | Neefjes, M. Housmans, B. A. C. van den Akker, G. G. H. van Rhijn, L. W. Welting, T. J. M. van der Kraan, P. M. |
author_facet | Neefjes, M. Housmans, B. A. C. van den Akker, G. G. H. van Rhijn, L. W. Welting, T. J. M. van der Kraan, P. M. |
author_sort | Neefjes, M. |
collection | PubMed |
description | Reporter gene assays are widely used to study cellular signaling and transcriptional activity. Few studies describe the use of reporter genes for studying cellular responses on complex body fluids, such as urine and blood. Selection of the optimal reporter gene is crucial for study outcome. Here, we compared the characteristics of five reporter genes (Firefly luciferase, stable- and unstable Nano luciferase, secretable Gaussia luciferase and Red Fluorescent Protein) to study complex body fluids. For this comparison, the NFκB Response Element (NFκB-RE) and Smad Binding Element (SBE) were identically cloned into the five different reporter vectors. Reporter characteristics were evaluated by kinetic and concentration–response measurements in SW1353 and HeLa cell lines. Finally, reporter compatibility with complex body fluids (fetal calf serum, knee joint synovial fluid and human serum) and inter-donor variation were evaluated. Red Fluorescent Protein demonstrated poor inducibility as a reporter gene and slow kinetics compared to luciferases. Intracellularly measured luciferases, such as Firefly luciferase and Nano luciferase, revealed good compatibility with complex body fluids. Secreted Gaussia luciferase appeared to be incompatible with complex body fluids, due to variability in inter-donor signal interference. Unstable Nano luciferase demonstrated clear inducibility, high sensitivity and compatibility with complex body fluids and therefore can be recommended for cellular signaling studies using complex body fluids. |
format | Online Article Text |
id | pubmed-7809208 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-78092082021-01-15 Reporter gene comparison demonstrates interference of complex body fluids with secreted luciferase activity Neefjes, M. Housmans, B. A. C. van den Akker, G. G. H. van Rhijn, L. W. Welting, T. J. M. van der Kraan, P. M. Sci Rep Article Reporter gene assays are widely used to study cellular signaling and transcriptional activity. Few studies describe the use of reporter genes for studying cellular responses on complex body fluids, such as urine and blood. Selection of the optimal reporter gene is crucial for study outcome. Here, we compared the characteristics of five reporter genes (Firefly luciferase, stable- and unstable Nano luciferase, secretable Gaussia luciferase and Red Fluorescent Protein) to study complex body fluids. For this comparison, the NFκB Response Element (NFκB-RE) and Smad Binding Element (SBE) were identically cloned into the five different reporter vectors. Reporter characteristics were evaluated by kinetic and concentration–response measurements in SW1353 and HeLa cell lines. Finally, reporter compatibility with complex body fluids (fetal calf serum, knee joint synovial fluid and human serum) and inter-donor variation were evaluated. Red Fluorescent Protein demonstrated poor inducibility as a reporter gene and slow kinetics compared to luciferases. Intracellularly measured luciferases, such as Firefly luciferase and Nano luciferase, revealed good compatibility with complex body fluids. Secreted Gaussia luciferase appeared to be incompatible with complex body fluids, due to variability in inter-donor signal interference. Unstable Nano luciferase demonstrated clear inducibility, high sensitivity and compatibility with complex body fluids and therefore can be recommended for cellular signaling studies using complex body fluids. Nature Publishing Group UK 2021-01-14 /pmc/articles/PMC7809208/ /pubmed/33446782 http://dx.doi.org/10.1038/s41598-020-80451-6 Text en © The Author(s) 2021 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Neefjes, M. Housmans, B. A. C. van den Akker, G. G. H. van Rhijn, L. W. Welting, T. J. M. van der Kraan, P. M. Reporter gene comparison demonstrates interference of complex body fluids with secreted luciferase activity |
title | Reporter gene comparison demonstrates interference of complex body fluids with secreted luciferase activity |
title_full | Reporter gene comparison demonstrates interference of complex body fluids with secreted luciferase activity |
title_fullStr | Reporter gene comparison demonstrates interference of complex body fluids with secreted luciferase activity |
title_full_unstemmed | Reporter gene comparison demonstrates interference of complex body fluids with secreted luciferase activity |
title_short | Reporter gene comparison demonstrates interference of complex body fluids with secreted luciferase activity |
title_sort | reporter gene comparison demonstrates interference of complex body fluids with secreted luciferase activity |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7809208/ https://www.ncbi.nlm.nih.gov/pubmed/33446782 http://dx.doi.org/10.1038/s41598-020-80451-6 |
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