Signed outside: A surface marker system for transgenic cytoplasmic proteins

V. Wohlgensinger, R. Seger, M. D. Ryan, J. Reichenbach, U. Siler*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)

Abstract

Chronic granulomatous disease is a primary immunodeficiency, comprising five molecular defects, characterized by an impaired respiratory burst activity of myeloid cells. We are currently developing a gene therapy vector for the p47phox-deficient form of chronic granulomatous disease. Classic intracellular immunostaining of the cytoplasmic p47phox transgene product, however, interferes with respiratory burst activity. In this study we report a new system for measuring p47phox expression: A single open reading frame encoding the surface marker protein ΔLNGFR (truncated low-affinity nerve growth factor receptor) linked to the p47phox transgene by the 2A oligopeptide coexpression technology. Translation generates two discrete products: p47phox localizing to the cytoplasm and ΔLNGFR-2A localizing to the cell surface. Six weeks after transplantation of transduced autologous hematopoietic stem cells into p47-/- mice, the intracellular p47phox fluorescence-activated cell sorting (FACS) signal intensities corresponded to surface ΔLNGFR staining in monocytes, B cells, T cells and Sca I bone marrow cells in vivo. The p47phox cleavage product restored nicotinamide adenine dinucleotide phosphate-oxidase activity in granulocytes differentiated from transduced p47phox/ murine hematopoietic stem cells ex vivo, in murine granulocytes/monocytes in vivo, and in transduced human monocyte derived macrophages from p47phox-deficient chronic granulomatous disease patients. In conclusion, this new marker system allows highly efficient, indirect detection of cytoplasmic transgene products by FACS surface staining.

Original languageEnglish
Pages (from-to)1193-1199
Number of pages7
JournalGene Therapy
Volume17
Issue number10
DOIs
Publication statusPublished - 1 Oct 2010

Keywords

  • δLNGFR
  • chronic granulomatous disease
  • marker protein
  • NADPH-oxidase
  • p47phox

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