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Related Experiment Video

Updated: Jun 13, 2026

Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks

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Published on: December 24, 2016

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

V Wohlgensinger¹, R Seger, M D Ryan

¹Division of Immunology/Hematology/BMT, University Children's Hospital Zürich, Steinwiesstrasse 75, Zürich, Switzerland.

|May 7, 2010

Summary

A novel gene therapy marker system enables indirect detection of p47phox expression in chronic granulomatous disease research. This method uses a surface marker to monitor cytoplasmic transgene delivery and restoration of myeloid cell function.

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Area of Science:

Immunology
Gene Therapy
Cell Biology

Background:

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by impaired myeloid cell respiratory burst activity.
The p47phox-deficient form of CGD requires effective gene therapy strategies.
Current intracellular staining methods for p47phox interfere with crucial cellular functions.

Purpose of the Study:

To develop a novel, non-interfering method for measuring p47phox transgene expression in CGD gene therapy.
To validate the efficacy of a new surface marker system for indirect detection of cytoplasmic p47phox.

Main Methods:

Utilized 2A oligopeptide coexpression technology to link the p47phox transgene with a truncated low-affinity nerve growth factor receptor (ΔLNGFR) surface marker.
Generated two discrete protein products: functional p47phox and cell-surface ΔLNGFR.
Transduced hematopoietic stem cells and assessed p47phox and ΔLNGFR expression in vivo and ex vivo using fluorescence-activated cell sorting (FACS).

Main Results:

Demonstrated a strong correlation between intracellular p47phox levels and surface ΔLNGFR staining in various immune cells in p47-/- mice.
Restored nicotinamide adenine dinucleotide phosphate-oxidase activity in granulocytes and macrophages from both murine models and human CGD patients.
Confirmed successful indirect detection of cytoplasmic transgene product via FACS surface staining.

Conclusions:

The developed ΔLNGFR-2A marker system provides a highly efficient and non-interfering method for monitoring p47phox gene therapy.
This approach enables reliable assessment of transgene expression and functional restoration in CGD.
The system holds significant potential for advancing gene therapy development and clinical applications for CGD.