Monitoring gene expression in vivo, noninvasively, is a critical issue in effective gene therapy systems. To date, there are no adequate molecular imaging techniques, which quantitatively monitor gene expression in vivo in skeletal development and repair. The aim of this study was to monitor gene expression in skeletal development and repair, using a real-time molecular imaging system, which quantitatively and noninvasively detects bioluminescence in vivo. Our experimental model consisted of transgenic mice harboring the luciferase marker gene under the regulation of the human osteocalcin (hOC) promoter. A new light detection cooled charge coupled device (CCCD) camera was applied to monitor luciferase expression. In vitro, mesenchymal stem cells (MSCs) isolated from bone marrow of transgenic mice exhibited hOC promoter regulation, detected by luciferase expression that correlated with their osteogenic differentiation. During development from 1 week to 1.5 years, transgenic mice exhibited transgene expression in a wide spectrum of skeletal organs, including calvaria, vertebra, tail, and limbs, reaching a peak at 1 week in most of the skeletal organs. In two skeletal repair models, bone fracture and marrow ablation, the noninvasive CCCD system revealed a peak of luciferase expression at 6 days postsurgery. All quantitative, noninvasive, real-time CCCD measurements correlated with a luciferase biochemical assay and luciferase immunohistochemistry, which demonstrated luciferase expression in hypertrophic chondrocytes and trabecular osteoblasts. Our studies show for the first time (1) the CCCD detection system is a reliable quantitative gene detection tool for the skeleton in vivo, (2) expression of luciferase regulated by the hOC promoter is significantly decreased with age in most skeletal sites, and (3) the dynamics of hOC regulation during mice skeletal development and repair in real time, quantitatively and noninvasively.

Monitoring the expression of therapeutic genes in targeted tissues in disease models is important to assessing the effectiveness of systems of gene therapy delivery. We applied a new light-detection cooled charged-coupled device (CCCD) camera for continuous in vivo assessment of commonly used gene therapy delivery systems (such as ex vivo manipulated cells, viral vectors, and naked DNA), without the need to kill animals. We examined a variety of criteria related to real-time monitoring of luciferase (luc) gene expression in tissues including bone, muscle, salivary glands, dermis, liver, peritoneum, testis, teeth, prostate, and bladder in living mice and rats. These criteria included determination of the efficiency of infection/transfection of various viral and nonviral delivery systems, promoter specificity, and visualization of luciferase activity, and of the ability of luciferin to reach various organs. The exposure time for detection of luc activity by the CCCD camera is relatively short (approximately 2 minutes) compared with the intensified CCD camera photon-counting method (approximately 15 minutes). Here we transduce a variety of vectors (such as viruses, transfected cells, and naked DNA) by various delivery methods, including electroporation, systemic injection of viruses, and tail-vein, high-velocity-high-volume administration of DNA plasmids. The location, intensity, and duration of luc expression in different organs were determined. The distribution of luciferin is most probably not a barrier for the detection of in vivo luciferase activity. We showed that the CCCD photon detection system is a simple, reproducible, and applicable method that enables the continuous monitoring of a gene delivery system in living animals.