Visualizing levels of osteoblast differentiation by a two‐color promoter‐GFP strategy: Type I collagen‐GFPcyan and osteocalcin‐GFPtpz

Abstract

A 3.9 kb DNA fragment of human osteocalcin promoter and 3.6 kb DNA fragment of the rat collagen type1a1 promoter linked with visually distinguishable GFP isomers, topaz and cyan, were used for multiplex analysis of osteoblast lineage progression. Three patterns of dual transgene expression can be appreciated in primary bone cell cultures derived from the transgenic mice and by histology of their corresponding bones. Our data support the interpretation that strong pOBCol3.6GFPcyan alone is found in newly formed osteoblasts, while strong pOBCol3.6GFPcyan and hOC-GFPtpz are present in osteoblasts actively making a new matrix. Osteoblasts expressing strong hOC-GFPtpz and weak pOBCol3.6GFPcyan are also present and may or may not be producing mineralized matrix. This multiplex approach reveals the heterogeneity within the mature osteoblast population that cannot be appreciated by current histological methods. It should be useful to identify and isolate populations of cells within an osteoblast lineage as they progress through stages of differentiation.

NF-κB Functions in Stromal Fibroblasts to Regulate Early Postnatal Muscle Development

“…4B), suggesting that NF-B activity derives from stromal fibroblasts in developing postnatal skeletal muscle. To confirm this notion, we screened for NF-B activity in muscles from reporter mice containing YFP under the control of the collagen type 1a promoter, which is activated in fibroblasts (31). Preparation of mononuclear cells from these mice at P8 and subsequent FACS sorting validated that NF-B activity, as measured by p-p65 protein, was indeed present in collagen 1a driving YFP ϩ cells (Fig.…”

“…, and collagen type 1a YFP mice were generated as previously described (27)(28)(29)(30)(31). iNOS Ϫ/Ϫ and wild type CD2 and C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME).…”

Mineralization and Bone Resorption Are Regulated by the Androgen Receptor in Male Mice

“…Furthermore, it has been recently identified that the mature osteoblast population is heterogeneous with distinct osteoblast gene expression profiles. (45) For example, a population of mature osteoblasts exist that express low levels of the 3.6-kb Col 1a1 promoter, which remain low when expression of the osteocalcin promoter is initiated. (45) It is possible, therefore, that a similar population of mature osteoblasts exist that express low levels of the Col2.3 promoter, which progress to become terminally differentiated mineralizing osteoblasts.…”

“…(45) For example, a population of mature osteoblasts exist that express low levels of the 3.6-kb Col 1a1 promoter, which remain low when expression of the osteocalcin promoter is initiated. (45) It is possible, therefore, that a similar population of mature osteoblasts exist that express low levels of the Col2.3 promoter, which progress to become terminally differentiated mineralizing osteoblasts. In the Col2.3-ARKOs, this low level of Col2.3 expression would give rise to a population of terminally differentiated mineralizing osteoblasts that retain AR expression, thereby resulting in the less severe bone phenotype of the Col2.3-ARKOs than expected.…”

In utero transplantation of adult bone marrow decreases perinatal lethality and rescues the bone phenotype in the knockin murine model for classical, dominant osteogenesis imperfecta

“…The bones were then decalcified in 14% ethylenediaminetetraacetic acid (EDTA)/PBS, pH 7.1, for 4 days 37 and incubated in 30% sucrose/PBS for 24 hours. All procedures were performed at 4°C in the dark.…”