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Visualizing Levels of Osteoblast Differentiation by a Two-Color Promoter-GFP Strategy: Type I Collagen-GFPcyan and Osteocalcin-GFPtpz

For understanding cellular and molecular mechanisms of osteoblast differentiation it is necessary to develop visual markers that reflect the progression of cells to full osteoblast differentiation in cell culture that should be readily transferable to in vivo models of bone formation. The purpose of this study is to show the feasibility of generating transgenic mice containing two promoter constructs driving distinguishable GFP isomers at different stages of osteoblast differentiation to identify cells at different levels of development both in cell culture and intact mouse bone. A 3.9 kb fragment of the human osteocalcin (hOC) promoter and a 3.6 kb fragment of the collagen promoter (Col3.6) linked with visually distinguishable GFP’ s, topaz and cyan, were used for multiplex analysis of osteoblast lineage progression. Transgenic mice for each construct, identified as pOBCol3.6GFPcyan and hOCGFPtpz, were individually characterized and then crossed. Temporal and spatial expression of both transgenes was analyzed in vitro and in vivo. In marrow stromal and neonatal calvarial cell cultures of crossed transgenic lines the pOBCol3.6GFPcyan low intensity positive cells appeared on day 7 and its expression continued as high intensity GFP positive cells that contributed to the formation of nodules. The hOCGFPtpz transgene expressing cells appeared at day 15 in nodules undergoing mineralization. In the mineralizing nodule we observed the activation of hOCGFPtpz in cells that were originally only cyan positive. The intensity of tpz in double GFP positive cells gradually increased as a concomitant decrease in cyan expression was detected. In the histological analysis, the double transgenics illustrate a spectrum of osteoblast differentiation. Cells expressing cyan are present in the primary spongiosa and extend a short distance along the cortical bone. Double expressing cells can be found in trabeculae in the secondary spongiosa and lower cortex and along the surface of cortical bone. The majority of cells on the surface of cortical and trabecular bone and osteocytes are only tpz positive. Thus a spectrum of osteoblast differentiation can be observed in bone nodules in primary culture and within bone. Because genetic disorders of bone may ultimately exert their effect by interfering with the orderly progression of progenitor to fully mature bone cell, this approach may be useful to identify the cellular consequence of a gene mutation as progenitors progress through stages of differentiation in cell culture and intact bone.

GFP ; osteocalcin promoter ; osteoblast differentiation ; type I collagen promoter ; primary osteoblast culture ; bone histology

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