Enhanced reporter gene assay for the detection of osteogenic differentiation.
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.
Ludwig Boltzmann Institute for Experimental and Clinical Traumatology/AUVA Research Center, The Austrian Cluster for Tissue Regeneration, European Institute of Excellence on Tissue Engineering and Regenerative Medicine Research (Expertissues EEIG), Vienna, Austria. georg.feichtinger@trauma.lbg.ac.at
Georg A
GA
Feichtinger
Tatjana J
TJ
Morton
Alice
A
Zimmermann
Daniela
D
Dopler
Asmita
A
Banerjee
Heinz
H
Redl
Martijn
M
van Griensven
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology; D002113:Calcification, Physiologic / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D005453:Fluorescence; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics*; D005821:Genetic Techniques; D006801:Humans; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D013194:Staining and Labeling; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C499469:recombinant human bone morphogenetic protein-2; D015675:Osteocalcin; D000469:Alkaline Phosphatase