Signed outside: a surface marker system for transgenic cytoplasmic proteins.

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.

• Authors: V Wohlgensinger, R Seger, M D Ryan, J Reichenbach, U Siler
• Journal: Gene therapy (Gene Ther) Vol. 17 Issue 10 Pg. 1193-9 (Oct 2010) ISSN: 1476-5462 [Electronic] England
• PMID: 20445581 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Biomarkers
• Receptors, Nerve Growth Factor
• NADPH Oxidases
• neutrophil cytosolic factor 1

Topics

• Animals
• Biomarkers (chemistry)
• Flow Cytometry
• Genetic Therapy
• Genetic Vectors (genetics)
• Granulomatous Disease, Chronic (genetics, metabolism, therapy)
• Hematopoietic Stem Cells (cytology, metabolism)
• Humans
• Mice
• NADPH Oxidases (genetics, metabolism)
• Receptors, Nerve Growth Factor (genetics)
• Transgenes (genetics)