A customized retroviral vector confers marker gene expression in osteoclast lineage cells

✍ Scribed by Daniel J. Selski; Denis R. Clohisy

• Publisher: John Wiley and Sons
• Year: 2006
• Tongue: English
• Weight: 352 KB
• Volume: 97
• Category: Article
• ISSN: 0730-2312
• DOI: 10.1002/jcb.20679

✦ Synopsis

Abstract

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 (γ‐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. J. Cell. Biochem. © 2005 Wiley‐Liss, Inc.