Use of Type I Collagen Green Fluorescent Protein Transgenes to Identify Subpopulations of Cells at Different Stages of the Osteoblast Lineage

Green fluorescent protein (GFP)‐expressing transgenic mice were produced containing a 3.6‐kilobase (kb; pOBCol3.6GFPtpz) and a 2.3‐kb (pOBCol2.3GFPemd) rat type I collagen (Col1a1) promoter fragment. The 3.6‐kb promoter directed strong expression of GFP messenger RNA (mRNA) to bone and isolated tail tendon and lower expression in nonosseous tissues. The 2.3‐kb promoter expressed the GFP mRNA in the bone and tail tendon with no detectable mRNA elsewhere. The pattern of fluorescence was evaluated in differentiating calvarial cell (mouse calvarial osteoblast cell [mCOB]) and in marrow stromal cell (MSC) cultures derived from the transgenic mice. The pOBCol3.6GFPtpz‐positive cells first appeared in spindle‐shaped cells before nodule formation and continued to show a strong signal in cells associated with bone nodules. pOBCol2.3GFPemd fluorescence first appeared in nodules undergoing mineralization. Histological analysis showed weaker pOBCol3.6GFPtpz‐positive fibroblastic cells in the periosteal layer and strongly positive osteoblastic cells lining endosteal and trabecular surfaces. In contrast, a pOBCol2.3GFPemd signal was limited to osteoblasts and osteocytes without detectable signal in periosteal fibroblasts. These findings suggest that Col1a1GFP transgenes are marking different subpopulations of cells during differentiation of skeletal osteoprogenitors. With the use of other promoters and color isomers of GFP, it should be possible to develop experimental protocols that can reflect the heterogeneity of cell differentiation in intact bone. In primary culture, this approach will afford isolation of subpopulations of these cells for molecular and cellular analysis.

[1]  R. Burgeson,et al.  Selective activation of the versican promoter by epithelial- mesenchymal interactions during hair follicle development. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[2]  M. Capecchi,et al.  Detection of targeted GFP-Hox gene fusions during mouse embryogenesis. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[3]  Joseph Zaia,et al.  Mesenchymal Stem Cell Surface Antigen SB‐10 Corresponds to Activated Leukocyte Cell Adhesion Molecule and Is Involved in Osteogenic Differentiation , 1998, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[4]  D. Prockop.  Marrow Stromal Cells as Stem Cells for Nonhematopoietic Tissues , 1997, Science.

[5]  H. Blau,et al.  Migration of myoblasts across basal lamina during skeletal muscle development , 1990, Nature.

[6]  D. Rowe,et al.  Col1a1‐Driven Transgenic Markers of Osteoblast Lineage Progression , 2001, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[7]  A. McMahon,et al.  Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis. , 2000, Development.

[8]  A. Fine,et al.  Live astrocytes visualized by green fluorescent protein in transgenic mice. , 1997, Developmental biology.

[9]  D. Rowe,et al.  COL1A1 Transgene Expression in Stably Transfected Osteoblastic Cells , 1997, The Journal of Biological Chemistry.

[10]  D. Rowe,et al.  Transgenic expression of COL1A1-chloramphenicol acetyltransferase fusion genes in bone: differential utilization of promoter elements in vivo and in cultured cells , 1993, Molecular and cellular biology.

[11]  C. A. Thomas,et al.  Molecular cloning. , 1977, Advances in pathobiology.

[12]  D. V. Cohn,et al.  Target cells in bone for parathormone and calcitonin are different: enrichment for each cell type by sequential digestion of mouse calvaria and selective adhesion to polymeric surfaces. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[13]  D. Rowe,et al.  Construction of DNA sequences complementary to rat alpha 1 and alpha 2 collagen mRNA and their use in studying the regulation of type I collagen synthesis by 1,25-dihydroxyvitamin D. , 1984, Biochemistry.

[14]  C. Cepko,et al.  Clonal analysis in the chicken retina reveals tangential dispersion of clonally related cells. , 1994, Developmental biology.

[15]  J. Aubin,et al.  The mature osteoblast phenotype is characterized by extensive plasticity. , 1997, Experimental cell research.

[16]  A. Faerman,et al.  Regulatory elements that control the lineage-specific expression of myoD. , 1992, Science.

[17]  P. Bianco,et al.  Multipotential cells in the bone marrow stroma: regulation in the context of organ physiology. , 1999, Critical reviews in eukaryotic gene expression.

[18]  S. Chiu,et al.  A novel P0 glycoprotein transgene activates expression of lacZ in myelin‐forming Schwann cells , 1999, The European journal of neuroscience.

[19]  C. Andressen,et al.  Cardiac specific expression of the green fluorescent protein during early murine embryonic development , 1998, FEBS letters.

[20]  J. Aubin,et al.  Advances in the osteoblast lineage. , 1998, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[21]  V. D’Agati,et al.  Expression of green fluorescent protein in the ureteric bud of transgenic mice: a new tool for the analysis of ureteric bud morphogenesis. , 1999, Developmental genetics.

[22]  Randall W. Smith,et al.  Col2‐GFP reporter marks chondrocyte lineage and chondrogenesis during mouse skeletal development , 2000, Developmental dynamics : an official publication of the American Association of Anatomists.

[23]  R. Oreffo,et al.  Skeletal progenitor cells and ageing human populations. , 1998, Clinical science.

[24]  J. Aubin,et al.  Simultaneous detection of multiple bone-related mRNAs and protein expression during osteoblast differentiation: polymerase chain reaction and immunocytochemical studies at the single cell level. , 1994, Developmental biology.

[25]  Tomoko Nakanishi,et al.  ‘Green mice’ as a source of ubiquitous green cells , 1997, FEBS letters.

[26]  K. Ozawa,et al.  Long-term tracking of murine hematopoietic cells transduced with a bicistronic retrovirus containing CD24 and EGFP genes , 2000, Gene Therapy.

[27]  B. Stillman,et al.  Cold Spring Harbor Laboratory , 1995, Molecular medicine.

[28]  G Cossu,et al.  Muscle regeneration by bone marrow-derived myogenic progenitors. , 1998, Science.

[29]  S. Gronthos,et al.  The STRO-1+ fraction of adult human bone marrow contains the osteogenic precursors. , 1994, Blood.

[30]  D. Rowe,et al.  Upstream regulatory elements necessary for expression of the rat COL1A1 promoter in transgenic mice , 1994, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[31]  M. Pittenger,et al.  Multilineage potential of adult human mesenchymal stem cells. , 1999, Science.

[32]  J. Aubin,et al.  Monoclonal antibodies as tools for studying the osteoblast lineage , 1996, Microscopy research and technique.

[33]  C. Kozak,et al.  Murine bone sialoprotein (BSP): cDNA cloning, mRNA expression, and genetic mapping , 1994, Mammalian Genome.

[34]  J. Aubin,et al.  Kinetics of osteoprogenitor proliferation and osteoblast differentiation in vitro , 1999, Journal of cellular biochemistry.

[35]  J. Rossert,et al.  Separate cis-acting DNA elements of the mouse pro-alpha 1(I) collagen promoter direct expression of reporter genes to different type I collagen-producing cells in transgenic mice , 1995, The Journal of cell biology.

[36]  S. Graves,et al.  Formation of mineralized nodules by bone derived cells in vitro: a model of bone formation? , 1993, American journal of medical genetics.

[37]  Thomas N. Sato,et al.  Universal GFP reporter for the study of vascular development , 2000, Genesis.

[38]  P. Bianco,et al.  CHAPTER 14 – Cellular Mechanisms of Age-Related Bone Loss , 1999 .

[39]  S. Bruder,et al.  Osteogenic differentiation of purified, culture‐expanded human mesenchymal stem cells in vitro , 1997, Journal of cellular biochemistry.

[40]  P. Bianco,et al.  Marrow stromal stem cells. , 2000, The Journal of clinical investigation.

[41]  V. Rosen,et al.  Isolation of the human gene for bone gla protein utilizing mouse and rat cDNA clones. , 1986, The EMBO journal.

[42]  Dieter Klein,et al.  Rapid and Sensitive Detection of Enhanced Green Fluorescent Protein Expression in Paraffin Sections by Confocal Laser Scanning Microscopy , 2000, The Histochemical Journal.

[43]  D. N. Landon,et al.  Angiopoietin-2 is a site-specific factor in differentiation of mouse renal vasculature. , 2000, Journal of the American Society of Nephrology : JASN.

[44]  S. Hughes,et al.  Replication-competent retrovirus vectors for the transfer and expression of gene cassettes in avian cells , 1991, Journal of virology.

[45]  S. Manolagas,et al.  Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. , 2000, Endocrine reviews.

[46]  S. Gronthos,et al.  Differential Cell Surface Expression of the STRO‐1 and Alkaline Phosphatase Antigens on Discrete Developmental Stages in Primary Cultures of Human Bone Cells , 1999, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[47]  D. Rowe,et al.  Identification of a TAAT-containing Motif Required for High Level Expression of the COL1A1 Promoter in Differentiated Osteoblasts of Transgenic Mice* , 1996, The Journal of Biological Chemistry.

[48]  J. Gordon,et al.  Use of transgenic mice to infer the biological properties of small intestinal stem cells and to examine the lineage relationships of their descendants. , 1991, Proceedings of the National Academy of Sciences of the United States of America.