Original Full Length ArticleBioluminescence imaging of bone formation using hairless osteocalcin-luciferase transgenic mice
Highlights
► In vivo bioluminescence imaging is useful in the study of bone development and regeneration. ► A hairless transgenic mouse line that expresses luciferase under the control of osteocalcin enhancer/promoter sequences was established. ► The hairless background enabled easy analysis of bone formation activity through bioluminescence in the entire mouse. ► We identified abrupt decrease in bone formation activity as the mice reached puberty and gradual further decreases with age. ► Retarded osteocalcin expression during fracture healing in aged mice, as compared with young mice, suggests age-related impairment of healing.
Introduction
Bone is a dynamic tissue that undergoes modeling during childhood and continuous remodeling throughout adulthood [1], [2]. Maintenance of bone mass and density is dependent on the balance between two processes: resorption of mineralized bone by osteoclasts and de novo bone formation by osteoblasts. An alteration in either or both of these processes causes a change in the bone density. Bone loss and the resultant skeletal structural weakening are associated with aging as a consequence of changes in hormone levels, bone cell differentiation and function [3]. This loss of bone may be especially debilitating in post-menopausal women, giving rise to osteoporosis and the consequential morbidity and mortality associated with increased incidence of fractures [2], [4]. While there is an analogous age-related skeletal deterioration in mice [5], [6], [7], additional data are needed to better describe age-dependent changes in bone structure and strength.
Osteocalcin is the most abundant noncollagenous protein expressed in bone, with its expression limited to cells of the osteoblast lineage, including mature osteoblasts, osteocytes, and hypertrophic chondrocytes [8], [9]. The location of osteocalcin at bone-forming surfaces [10] and the increased bone mineralization observed in osteocalcin gene knockout mice [11] support a role for osteocalcin in the suppression of bone mineralization. Alternatively, osteocalcin has been suggested to increase bone resorption through osteoclast recruitment [10], [12]. Osteocalcin also has features of a hormone, as it is synthesized as a prepromolecule and secreted in the general circulation [9]. Further, osteocalcin acts as a regulator of insulin in the pancreas, adiponectin in adipocytes [13], and testosterone synthesis in male germ line cells [14]. Thus, osteocalcin is an important molecule that functions not only in bone, but also in energy metabolism and reproduction.
In recent years, several highly sensitive imaging technologies have been developed to detect and quantitate in vivo fluorescence and luminescence, without sacrificing animals [15], [16]. These technologies are important in developmental studies, in gene therapy systems, and for the purpose of revealing the spatiotemporal patterns and expression intensity of target genes. Previous studies have employed a segment of the human osteocalcin (OC) promoter to analyze OC promoter regulation in mice [17], [18], [19]. Kesterson et al. [18] reported the use of transgenic mice harboring this 3.9 kb promoter sequence and 10 bp of 5′-untranslated sequence fused to a chloramphenicol acetyltransferase reporter gene, and noted its expression in bone-associated tissues. Utilizing the same promoter, Iris et al. and Bilic-Curcic et al. also demonstrated that luciferase and GFP, respectively, were expressed in a wide spectrum of skeletal organs, increasing several days after bone fracture [20], [21], [22]. Using in vivo bioluminescent imaging, the human OC promoter was also shown to exhibit a periodicity of approximately 24 h in multiple skeletal sites [23].
In this study, we produced a transgenic mouse line expressing luciferase under the control of 10 kb osteocalcin enhancer/promoter sequence. This line was backcrossed with a hairless mouse line to enable us to monitor bone formation during growth, aging, and fracture repair using in vivo imaging, without sacrificing the mouse. The bioluminescence monitored during fracture healing showed delayed osteocalcin expression in aged mice as compared with young mice, suggesting an age-related impairment during fracture repair. Our bioluminescence imaging system may offer a powerful tool for the non-invasive tracking of bone formation activity in vivo during bone development and regeneration.
Section snippets
Plasmid construction
A pBSI-I plasmid was a kind gift from Dr. Mitsuo Oshimura (Tottori University, Tottori, Japan). This plasmid contained two copies of the 1.2 kb 5′ β-globin element derived from the chicken constitutive hypersensitive site (5′HS4) responsible for an insulating effect. pGL3-Basic and pKO Scrambler V907 were purchased from Promega (Madison, WI, USA) and Lexicon Genetics (Woodlands, TX, USA), respectively. The pGL3-Basic-AB retrieval vector was generated by inserting a 405 bp NheI/EcoRI-homologous
Construction of a plasmid expressing luciferase under the control of human osteocalcin
A human osteocalcin gene enhancer/promoter fragment (OC) containing approximately 10 kb of up-stream sequence and 60 bp of 5′-untranslated sequence was retrieved from a BAC clone utilizing the defective prophage λ-Red recombineering system [24]. The fragment contains a 1α,25-dihydroxyvitamin D3 (referred to as D3)-responsible element (VDRE), a GAGA DNA motif, suggested to control D3 responsiveness of the rat osteocalcin gene [29], [30], and a TATA box. The osteocalcin translation start codon,
Discussion
Animal models are commonly used in the study of skeletal biology and serve as useful tools to delineate mechanisms underlying bone loss and skeletal fragility. In this study, we generated transgenic mice using the pOC-Luc construct, which expresses luciferase under the control of the 10 kb human osteocalcin enhancer/promoter sequences (Fig. 1A), to maximize our chances of identifying important regulatory regions of human osteocalcin. All three of the transgenic mouse lines established with
Conclusions
We have shown that a 10 kb fragment of the human osteocalcin enhancer/promoter sequences responds to D3 and shows retarded expression in bone healing with aging. These results indicate that this fragment contains sufficient information to target a heterologous reporter gene to bone-associated tissues in mice. Our in vivo imaging system may contribute to the therapy and prevention of various bone metabolic disorders by monitoring the process of bone formation. Furthermore, our mouse system offers
Acknowledgments
The authors wish to thank Dr. Naohiro Hori, Ms. Mayu Yasunaga, and our laboratory members for technical assistance and valuable discussions. This study was supported in part by a City area program (basic stage) a Regional Innovation Cluster Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT).
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