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Signed outside: a surface marker system for transgenic cytoplasmic proteins

Gene Therapy volume 17, pages 1193–1199 (2010)Cite this article

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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.

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Acknowledgements

We thank Manuel Grez, Georg Speyer Research Institute, Frankfurt, Germany for the generous gift of vector backbones and helpful discussions. This study was supported by ‘Forschungskredit der Universität Zürich 2005’ and Novartis Stiftung für Medizinisch-Biologische Forschung and Swiss National Science Foundation (Grant no. 320000–121983) to US.

Authorship contribution: VW performed experiments; US, RS and MDR designed the research; US, JR and VW analyzed results and made the figures; US, MDR, RS and JR wrote the paper.

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Authors and Affiliations

  1. Division of Immunology/Hematology/BMT, University Children's Hospital Zürich, Zürich, Switzerland

    V Wohlgensinger, R Seger, J Reichenbach & U Siler

  2. Centre for Biomolecular Sciences, School of Biology, University of St Andrews, St Andrews, UK

    M D Ryan

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  1. V Wohlgensinger
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  2. R Seger
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  3. M D Ryan
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  4. J Reichenbach
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  5. U Siler
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Correspondence to U Siler.

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Wohlgensinger, V., Seger, R., Ryan, M. et al. Signed outside: a surface marker system for transgenic cytoplasmic proteins. Gene Ther 17, 1193–1199 (2010). https://doi.org/10.1038/gt.2010.73

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