Construction of a Luciferase Reporter System to Monitor Osteogenic Differentiation of Mesenchymal Stem Cells by Using a Mammalian Artificial Chromosome Vector.
Year: 2015
Type: article
Abstract: BACKGROUND: Mesenchymal stem cells (MSCs) hold promise for application in adult stem cell-mediated regenerative medicine in bone remodeling and fracture repair. MSCs in vitro can be directed to osteogenic lineage by dexamethasone (DEX); however, the use of DEX is not practical in clinical settings because of adverse side effects such as glucocorticoid-induced osteoporosis. For identifying substances that facilitate osteogenesis, a monitoring system, which detects the osteogenic differentiation stage of MSCs accurately and easily, is required. METHODS: By focusing on the human osteocalcin (OC) gene whose expression profile is described along with osteogenic differentiation, we constructed the luciferase (Luc) reporter gene driven by the enhancer/promoter sequence of the human OC gene (OC-Luc) utilizing a mammalian artificial chromosome. Mammalian artificial chromosome is a suitable platform for loading reporter constructs, because of its stable episomal maintenance in host cells, transferability into any cell and assurance of long-term physiological transgene expression. We loaded the OC-Luc on a mammalian artificial chromosome vector engineered from mouse chromosome (designated as mouse artificial chromosome, MAC) in Chinese hamster ovary cells (OC-Luc/MAC) and transferred this into human MSC cells via chromosome transfer. RESULTS: OC-Luc/MAC in human MSC cells are responsive to positive and negative stimulation by 1 alpha,25-dihydroxyvitamin D3 and DEX in differentiation stage of MSCs to osteoblasts, reflecting the manner of physiological expression. CONCLUSION: The OC-Luc/MAC reporter system may contribute not only to monitoring the osteogenic differentiation stage from MSC but also to identify novel osteogenic drugs.
Source: PubMed
Authors: Takashi Narai, Motonobu Katoh, Toshiaki Inoue, Makoto Taniguchi, Kanako Kazuki, Yasuhiro Kazuki, Kenzo Sato, Isamu Kodani, Kazuo Ryoke, Mitsuo Oshimura
Institutions: Tottori University, Chromosome Engineering Research Center
Language: English
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2110316545