A customized retroviral vector confers marker gene expression in osteoclast lineage cells.
Osteoclasts play a seminal role in many skeletal diseases and therefore are candidates for cell-based gene delivery systems to treat disorders of bone. As an initial step toward developing osteoclast-mediated gene delivery systems, we have made and analyzed a customized Molony-Murine leukemia virus (MMLV)-based retroviral vector containing elements of the osteoclast-specific tartrate-resistant acid phosphatase (TRAP) gene. RAW 264.7 cells were transduced with the customized vector (E3) and differentiated along macrophage or osteoclast lineages. E3 contained a truncated form of the human nerve growth factor receptor (NGFR) as a reporter gene. NGFR expression increased with RANK-ligand (RANK-L) treatment but not with macrophage (gamma-IFN/LPS treatment) differentiation. Enhanced NGFR expression peaked 48 h after RANK-L treatment. Electrophoretic mobility shift assays (EMSA) analysis of the TRAP gene regulatory elements in E3 identified a single 27 bp DNA probe, which specifically bound protein from RANK-L-treated cells. DNA sequence revealed AP-1 binding sites, and analysis with mutant probes implied that the sites were functional. EMSA supershift analysis identified Fos protein interacting with the 27 bp probe. In summary, insertion of sequence -962 to -868 from the TRAP gene into the U3 region of the MMLV LTR confers RANK-L induced retroviral gene expression via Fos family protein interaction at AP-1 sites.
Selski
Daniel J
DJ
Department of Orthopaedic Surgery, The University of Minnesota, 420 Delaware Street, Minneapolis, MN 55455, USA.
Clohisy
Denis R
DR
D000135:Acid Phosphatase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins; D002454:Cell Differentiation; D019070:Cell Lineage; D002477:Cells; D024202:Electrophoretic Mobility Shift Assay; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D008562:Membrane Glycoproteins; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D016761:Oncogene Proteins v-fos; D010010:Osteoclasts / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism; D020218:Response Elements; D012190:Retroviridae / Q000235:genetics*; D000071681:Tartrate-Resistant Acid Phosphatase; D018808:Transcription Factor AP-1 / Q000378:metabolism
D002352:Carrier Proteins; D007527:Isoenzymes; D008562:Membrane Glycoproteins; D016761:Oncogene Proteins v-fos; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D017475:Receptors, Nerve Growth Factor; C505686:TNFRSF11A protein, human; C505688:TNFSF11 protein, human; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; D018808:Transcription Factor AP-1; D000135:Acid Phosphatase; C000605511:Acp5 protein, mouse; D000071681:Tartrate-Resistant Acid Phosphatase