Enhanced reporter gene assay for the detection of osteogenic differentiation
| Autor: | Martijn van Griensven, Alice Zimmermann, Tatjana J. Morton, Daniela Dopler, Heinz Redl, Asmita Banerjee, Georg A. Feichtinger |
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| Rok vydání: | 2010 |
| Předmět: |
Physiologic/drug effects
Yellow fluorescent protein Gene Expression Regulation/drug effects Medicine (miscellaneous) Bone Morphogenetic Protein 2 Mice Genes Reporter Osteogenesis Transforming Growth Factor beta Recombinant Proteins/pharmacology Cells Cultured Plasmids/genetics Cultured biology Cell Differentiation Recombinant Proteins Osteogenesis/drug effects Genetic Techniques Bone Morphogenetic Proteins Bioreporter Transforming Growth Factor beta/pharmacology C2C12 Plasmids Cells Osteocalcin Biomedical Engineering Bioengineering Bone morphogenetic protein Transfection Bone morphogenetic protein 2 Fluorescence Calcification Reporter/genetics Calcification Physiologic Animals Humans Luciferase RNA Messenger Enhancer Reporter gene Osteoblasts Bone Morphogenetic Proteins/pharmacology Staining and Labeling Osteoblasts/cytology Alkaline Phosphatase Molecular biology Genes Gene Expression Regulation Alkaline Phosphatase/metabolism Cell Differentiation/drug effects biology.protein RNA Messenger/genetics Osteocalcin/genetics |
| Zdroj: | Tissue engineering. Part C, Methods. 17(4) |
| ISSN: | 1937-3392 1937-3384 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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