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
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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