title: Construction of a Luciferase Reporter System to Monitor Osteogenic Differentiation of Mesenchymal Stem Cells by Using a Mammalian Artificial Chromosome Vector.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Division of Oral and Maxillofacial Biopathological Surgery, Department of Medicine of Sensory and Motor Organs, School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8504, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Narai
authors.affiliation: †Division of Human Genome Science, Department of Molecular and Cellular Biology, School of Life Sciences, Tottori University Faculty of Medicine, Yonago 683-8503, Japan ; §Chromosome Engineering Research Center, Tottori University, Yonago 683-8503, Japan.
authors.forename: Motonobu
authors.initials: M
authors.lastname: Katoh
authors.affiliation: †Division of Human Genome Science, Department of Molecular and Cellular Biology, School of Life Sciences, Tottori University Faculty of Medicine, Yonago 683-8503, Japan ; §Chromosome Engineering Research Center, Tottori University, Yonago 683-8503, Japan.
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Inoue
authors.affiliation: ‡Division of Molecular Biology, Department of Molecular and Cellular Biology, School of Life Sciences, Tottori University Faculty of Medicine, Yonago 683-8503, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Taniguchi
authors.affiliation: §Chromosome Engineering Research Center, Tottori University, Yonago 683-8503, Japan ; ‖Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Tottori University, Yonago, Tottori 683-8503, Japan.
authors.forename: Kanako
authors.initials: K
authors.lastname: Kazuki
authors.affiliation: §Chromosome Engineering Research Center, Tottori University, Yonago 683-8503, Japan ; ‖Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Tottori University, Yonago, Tottori 683-8503, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kazuki
authors.affiliation: ‡Division of Molecular Biology, Department of Molecular and Cellular Biology, School of Life Sciences, Tottori University Faculty of Medicine, Yonago 683-8503, Japan.
authors.forename: Kenzo
authors.initials: K
authors.lastname: Sato
authors.affiliation: Division of Oral and Maxillofacial Biopathological Surgery, Department of Medicine of Sensory and Motor Organs, School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8504, Japan.
authors.forename: Isamu
authors.initials: I
authors.lastname: Kodani
authors.affiliation: Division of Oral and Maxillofacial Biopathological Surgery, Department of Medicine of Sensory and Motor Organs, School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8504, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Ryoke
authors.affiliation: §Chromosome Engineering Research Center, Tottori University, Yonago 683-8503, Japan.
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Oshimura
keywords: mammalian artificial chromosome; mesenchymal stem cells; osteocalcin; osteogenic differentiation; reporter assay