Abstract
Specific mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), the most common autosomal recessive fatal genetic disease of Caucasians, result in the loss of epithelial cell adenosine 3',5'-cyclic-monophosphate (cAMP)-stimulated Cl- conductance. We show that the influx of a fluorescent dye, dihydrorhodamine 6G (dR6G), is increased in cells expressing human CFTR after retrovirus- and adenovirus-mediated gene transfer. dR6G influx is stimulated by cAMP and is inhibited by antagonists of cAMP action. Dye uptake is ATP-dependent and inhibited by Cl- removal or the addition of 10 mM SCN-. Increased staining is associated with functional activation of CFTR Cl- permeability. dR6G staining enables both the fluorescent assessment of CFTR function and the identification of successfully corrected cells after gene therapy.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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3T3 Cells
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ATP Binding Cassette Transporter, Subfamily B, Member 1 / biosynthesis
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Adenoviridae
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Animals
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Biological Transport
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Bronchi
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Cell Line
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Chlorides / metabolism*
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Cyclic AMP / analogs & derivatives
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Cyclic AMP / pharmacology
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Cyclic AMP / physiology*
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Cystic Fibrosis / genetics
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Cystic Fibrosis / metabolism
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Cystic Fibrosis Transmembrane Conductance Regulator / biosynthesis*
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Cystic Fibrosis Transmembrane Conductance Regulator / metabolism
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Endocytosis
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Epithelium
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Fluorescent Dyes / pharmacokinetics*
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Gene Expression*
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Genetic Vectors
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Humans
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Kinetics
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Mice
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Microscopy, Fluorescence
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / metabolism
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Retroviridae
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Rhodamines / pharmacokinetics
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Staining and Labeling
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Thionucleotides / pharmacology
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Transfection
Substances
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ATP Binding Cassette Transporter, Subfamily B, Member 1
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CFTR protein, human
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Chlorides
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Fluorescent Dyes
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Recombinant Proteins
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Rhodamines
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Thionucleotides
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dihydrorhodamine 6G
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Cystic Fibrosis Transmembrane Conductance Regulator
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adenosine-3',5'-cyclic phosphorothioate
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Cyclic AMP