© 1995 Oxford University Press
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The introduction of two silent mutations into a CFTR cDNA construct allows improved detection of exogenous mRNA in gene transfer experiments
Department of Biochemistry and Molecular Genetics, St. Mary's Hospital Medical School London, W2 1PG 1Ion Transport Unit, National Heart and Lung Institute London, SW3 6LR, UK 2Department of Pharmacology, University of Pittsburgh Pittsburgh, PA 15261, USA
*To whom correspondence should be addressed
Received May 3, 1995; Revised June 21, 1995; Accepted June 21, 1995
Phase one clinical trials for gene therapy of cystic fibrosis are in progress using either liposomes or adenoviral vectors for CFTR gene transfer to epithelial cells in the airways. In addition to electrophysiological measurements, expression of vector CFTR is usually assessed by RT-PCR. We have developed a CFTR-expression vector, pCFAS, that simplifies the distinction of transgene-derived CFTR mRNA from endogenous mRNA. Two point mutations were introduced into CFTR cDNA which eliminated a Sphl restriction site and created a new, unique Agel restriction site. Neither mutation altered the predicted amino acid sequence of the protein. Restriction digestion of RT-PCR products from cells transfected with pCFAS allowed the differentiation of transgene and endogenous CFTR transcripts. To verify function of the mutated CFTR, the plasmid was transferred into freshly obtained nasal epithelial cells from CF patients ex vivo using cationic liposomes. Fluorescence microscopy using the halide-sensitive fluorophore SPQ demonstrated restoration of cAMP-mediated Ci-secretion. This plasmid will be useful for CFTR gene transfer studies in vitro and in vivo.