An immunocytochemical assay to detect human CFTR expression following gene transfer

Medical Genetics, School of Molecular and Clinical Medicine, University of Edinburgh, Western General Hospital, Edinburgh EH42XU, UK.
Molecular and Cellular Probes (Impact Factor: 1.85). 08/2009; 23(6):272-80. DOI: 10.1016/j.mcp.2009.07.001
Source: PubMed


To assess gene therapy treatment for cystic fibrosis (CF) in clinical trials it is essential to develop robust assays that can accurately detect transgene expression in human airway epithelial cells. Our aim was to develop a reproducible immunocytochemical assay for human CFTR protein which can measure both endogenous CFTR levels and augmented CFTR expression after gene delivery.
We characterised an antibody (G449) which satisfied the criteria for use in clinical trials. We optimised our immunocytochemistry method and identified G449 dilutions at which endogenous CFTR levels were negligible in CF samples, thus enhancing detection of transgenic CFTR protein. After developing a transfection technique for brushed human nasal epithelial cells, we transfected non-CF and CF cells with a clinically relevant CpG-free plasmid encoding human CFTR.
The optimised immunocytochemistry method gave improved discrimination between CF and non-CF samples. Transfection of a CFTR expression vector into primary nasal epithelial cells resulted in detectable RNA and protein expression. CFTR protein was present in 0.05-10% of non-CF cells and 0.02-0.8% of CF cells.
We have developed a sensitive, clinically relevant immunocytochemical assay for CFTR protein and have used it to detect transgene-expressed CFTR in transfected human primary airway epithelial cells.

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    • "To aid identification and counting we co-stained the cytoplasm of the cells with IRF-1, a transcription factor that is localised throughout the cytoplasm of epithelial cells, as well as DAPI to stain the nucleus. Consistent with previous results using the MATG1061 antibody in freshly isolated nasal and bronchial epithelia cells [3], [5], [21] and freshly excised human tissue [7], [22] we found that the majority of non-CF cells showing a strong CFTR signal were TCE cells and CFTR appeared as a discrete band at the apical pole (Fig. 1B). Further analysis showed that the apical CFTR band was located just below the cilia (Fig. 1C) and that staining was absent in isotype matched immunoglobulins negative controls (Fig. 1D). "
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