Feichtinger, G orcid.org/0000-0002-7724-3006, Morton, TJ, Zimmermann, A et al. (4 more authors) (2011) Enhanced reporter gene assay for the detection of osteogenic differentiation. Tissue Engineering Part C: Methods, 17 (4). pp. 401-410. ISSN 1937-3384
Abstract
Detection of osteogenic differentiation is crucial for bone tissue engineering. Despite established standard end point assays, there is increasing demand for methods allowing noninvasive kinetic differentiation monitoring. Reporter gene assays employing tissue-specific promoters and suitable reporter genes fulfill these requirements. Many promoters, however, exhibit only weak cis-activating potential, thus limiting their application to generate sensitive reporter gene assays. Therefore, the aim of this study was to design a reporter gene assay employing elements of the murine osteocalcin promoter coupled to a viral enhancer for signal amplification. Additionally, the system's practicability was enhanced by introducing a secreted luciferase as a quantifiable reporter gene. The constructs were tested in C2C12 cells stimulated with recombinant human bone morphogenetic protein 2 for osteogenic differentiation in two-dimensional and three-dimensional culture. Osteogenic differentiation was confirmed by standard assays for osteogenesis. The reporter gene signal was detected through a secreted luciferase or fluorescence microscopy for enhanced yellow fluorescent protein. The constructs exhibited strong activation upon treatment with recombinant human bone morphogenetic protein 2. Weak background expression was observable in negative controls, attributed to the pan-active viral enhancer. In conclusion, a novel enhancer/tissue-specific promoter combination allows specific signal-amplified, kinetic monitoring of osteogenic differentiation in a nonsample-destructive manner.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2011, Mary Ann Liebert, Inc. This is an author produced version of an article published in Tissue Engineering Part C: Methods. Uploaded in accordance with the publisher's self-archiving policy. |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Medicine and Health (Leeds) > School of Dentistry (Leeds) > Oral Biology (Leeds) |
Depositing User: | Symplectic Publications |
Date Deposited: | 10 Sep 2019 08:25 |
Last Modified: | 10 Sep 2019 08:25 |
Published Version: | https://www.liebertpub.com/doi/10.1089/ten.tec.201... |
Status: | Published |
Publisher: | Mary Ann Liebert, Inc |
Identification Number: | 10.1089/ten.tec.2010.0095 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:96174 |