Document Detail

The introduction of two silent mutations into a CFTR cDNA construct allows improved detection of exogenous mRNA in gene transfer experiments.
MedLine Citation:
PMID:  8541845     Owner:  NLM     Status:  MEDLINE    
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 SphI restriction site and created a new, unique AgeI 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 Cl- secretion. This plasmid will be useful for CFTR gene transfer studies in vitro and in vivo.
S L Hart; E Mayall; M Stern; F M Munkonge; A Frost; L Huang; M Vasilliou; R Williamson; E W Alton; C Coutelle
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Human molecular genetics     Volume:  4     ISSN:  0964-6906     ISO Abbreviation:  Hum. Mol. Genet.     Publication Date:  1995 Sep 
Date Detail:
Created Date:  1996-02-14     Completed Date:  1996-02-14     Revised Date:  2009-09-29    
Medline Journal Info:
Nlm Unique ID:  9208958     Medline TA:  Hum Mol Genet     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  1597-602     Citation Subset:  IM    
Department of Biochemistry and Molecular Genetics, St. Mary's Hospital Medical School, London, UK.
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MeSH Terms
Base Sequence
Cell Line
Chlorides / metabolism
Cystic Fibrosis / metabolism
Cystic Fibrosis Transmembrane Conductance Regulator / genetics*
DNA, Complementary
Forskolin / pharmacology
Gene Transfer Techniques*
Genetic Vectors*
Ionomycin / pharmacology
Molecular Sequence Data
Nasal Mucosa / drug effects,  metabolism
Transcription, Genetic
Grant Support
//Wellcome Trust
Reg. No./Substance:
0/CFTR protein, human; 0/Chlorides; 0/DNA, Complementary; 126880-72-6/Cystic Fibrosis Transmembrane Conductance Regulator; 56092-81-0/Ionomycin; 66428-89-5/Forskolin

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