Signed outside: a surface marker system for transgenic cytoplasmic proteins

摘要: 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 gene therapy vector for the p47phox-deficient form chronic disease. Classic intracellular immunostaining cytoplasmic p47phox transgene product, however, interferes with activity. In this study we report new system measuring expression: A single open reading frame encoding surface marker protein ΔLNGFR (truncated low-affinity nerve growth factor receptor) linked to 2A oligopeptide coexpression technology. Translation generates two discrete products: localizing cytoplasm and 'ΔLNGFR-2A' cell surface. Six weeks after transplantation transduced autologous hematopoietic stem cells into p47-/- mice, fluorescence-activated sorting (FACS) signal intensities corresponded staining in monocytes, B cells, T Sca I+ bone marrow vivo. The cleavage product restored nicotinamide adenine dinucleotide phosphate-oxidase granulocytes differentiated from p47phox-/- murine ex vivo, granulocytes/monocytes human monocyte derived macrophages patients. conclusion, allows highly efficient, indirect detection products FACS staining.