USE OF A MEMBRANE POTENTIAL-SENSITIVE PROBE TO ASSESS BIOLOGICAL EXPRESSION OF THE CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR

Cystic fibrosis is caused by defects in a chloride-transporting protei n termed cystic fibrosis transmembrane conductance regulator (CFTR), T his study presents an innovative procedure to evaluate expression of f unctional CFTR, The technique uses the potential-sensitive probe bis-( 1,3-diethylthiobarbituric acid) trimethine oxonol or DiSBAC(2)(3), by single-cell fluorescence imaging. The DiSBAC(2)(3) method was first va lidated on the mouse mammary tumor cell line C127, stably expressing w ild-type CFTR, Activation of protein kinase A by the cAMP-permeable an alogue 8-Br-cAMP induced cell membrane depolarization consistent with expression of wild-type CFTR, The DiSBAC(2)(3) method is quick, simple , and reproducible, and does not require invasive cell loading procedu res. The system was then applied to the cell model of the human lung t umor cell line A549, in which exogenous CFTR was expressed by infectin g with the replication-deficient recombinant adenovirus AdCFTR, DiSBAC (2)(3) was able to detect the fraction of cells in which the expressio n of CFTR protein was confirmed by immunocytochemistry, The DiSBAC(2)( 3) probe was also used in human nasal respiratory cells cultured in vi tro, in which it efficiently discriminated between endogenous CFTR in normal and CF cells, Functional evaluation of CFTR function by the des cribed method can be a useful tool to detect the expression of the CF gene transferred by adenoviral vectors for use in gene therapy trials.