Reporter gene comparison demonstrates interference of complex body fluids with secreted luciferase activity

Abstract 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....

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Autores principales:	M. Neefjes, B. A. C. Housmans, G. G. H. van den Akker, L. W. van Rhijn, T. J. M. Welting, P. M. van der Kraan
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/17df234e8c6e430bae3c36e4f64af2bf
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spelling	oai:doaj.org-article:17df234e8c6e430bae3c36e4f64af2bf2021-12-02T14:12:46ZReporter gene comparison demonstrates interference of complex body fluids with secreted luciferase activity10.1038/s41598-020-80451-62045-2322https://doaj.org/article/17df234e8c6e430bae3c36e4f64af2bf2021-01-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-80451-6https://doaj.org/toc/2045-2322Abstract 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.M. NeefjesB. A. C. HousmansG. G. H. van den AkkerL. W. van RhijnT. J. M. WeltingP. M. van der KraanNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-13 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q M. Neefjes B. A. C. Housmans G. G. H. van den Akker L. W. van Rhijn T. J. M. Welting P. M. van der Kraan Reporter gene comparison demonstrates interference of complex body fluids with secreted luciferase activity
description	Abstract 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	article
author	M. Neefjes B. A. C. Housmans G. G. H. van den Akker L. W. van Rhijn T. J. M. Welting P. M. van der Kraan
author_facet	M. Neefjes B. A. C. Housmans G. G. H. van den Akker L. W. van Rhijn T. J. M. Welting P. M. van der Kraan
author_sort	M. Neefjes
title	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_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_sort	reporter gene comparison demonstrates interference of complex body fluids with secreted luciferase activity
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/17df234e8c6e430bae3c36e4f64af2bf
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Reporter gene comparison demonstrates interference of complex body fluids with secreted luciferase activity

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