dihydrorhodamine-6g and Cystic-Fibrosis

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.

Topics: 3T3 Cells; Adenoviridae; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Transport; Bronchi; Cell Line; Chlorides; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Endocytosis; Epithelium; Fluorescent Dyes; Gene Expression; Genetic Vectors; Humans; Kinetics; Mice; Microscopy, Fluorescence; Recombinant Proteins; Retroviridae; Rhodamines; Staining and Labeling; Thionucleotides; Transfection