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

Daniel J. Selski, Denis R Clohisy

Research output: Contribution to journalArticle

1 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)641-650
Number of pages10
JournalJournal of Cellular Biochemistry
Volume97
Issue number3
DOIs
StatePublished - Feb 15 2006

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Keywords

  • Acp5
  • Gene therapy
  • RAW 264.7
  • Retrovirus

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