title: 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.
authors.affiliation: Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut.
authors.forename: I
authors.initials: I
authors.lastname: Bilic-Curcic
authors.forename: M
authors.initials: M
authors.lastname: Kronenberg
authors.forename: X
authors.initials: X
authors.lastname: Jiang
authors.forename: J
authors.initials: J
authors.lastname: Bellizzi
authors.forename: M
authors.initials: M
authors.lastname: Mina
authors.forename: I
authors.initials: I
authors.lastname: Marijanovic
authors.forename: E M
authors.initials: EM
authors.lastname: Gardiner
authors.forename: D W
authors.initials: DW
authors.lastname: Rowe
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics*; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics; D006801:Humans; D049408:Luminescent Agents / Q000032:analysis*; D051379:Mice; D008822:Mice, Transgenic; D010006:Osteoblasts / Q000737:chemistry* / Q000166:cytology*; D015675:Osteocalcin / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D051381:Rats; D019076:Transgenes
chemical_list: D024042:Collagen Type I; D049408:Luminescent Agents; D015675:Osteocalcin; D049452:Green Fluorescent Proteins