Use of a membrane potential-sensitive probe to assess biological expression of the cystic fibrosis transmembrane conductance regulator

Human Gene Therapy
M RenierG Cabrini

Abstract

Cystic fibrosis is caused by defects in a chloride-transporting protein termed cystic fibrosis transmembrane conductance regulator (CFTR). This study presents an innovative procedure to evaluate expression of functional CFTR. The technique uses the potential-sensitive probe bis-(1,3-diethylthiobarbituric acid) trimethine oxonol or DiSBAC2(3), by single-cell fluorescence imaging. The DiSBAC2(3) method was first validated on the mouse mammary tumor cell line C127, stably expressing wild-type CFTR. Activation of protein kinase A by the cAMP-permeable analogue 8-Br-cAMP induced cell membrane depolarization consistent with expression of wild-type CFTR. The DiSBAC2(3) method is quick, simple, and reproducible, and does not require invasive cell loading procedures. The system was then applied to the cell model of the human lung tumor cell line A549, in which exogenous CFTR was expressed by infecting with the replication-deficient recombinant adenovirus AdCFTR. DiSBAC2(3) was able to detect the fraction of cells in which the expression of CFTR protein was confirmed by immunocytochemistry. The DiSBAC2(3) probe was also used in human nasal respiratory cells cultured in vitro, in which it efficiently discriminated between endogenous CFTR ...Continue Reading

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Citations

Jan 2, 2007·American Journal of Respiratory Cell and Molecular Biology·Maria Cristina DechecchiGiulio Cabrini
Jul 6, 2013·Pulmonary Pharmacology & Therapeutics·Caroline NorezClarisse Vandebrouck
Jul 5, 2005·Expert Opinion on Investigational Drugs·A Pavirani
Jun 23, 2006·European Journal of Immunology·Shibichakravarthy KannanMin Wu
Sep 27, 2008·Journal of Cystic Fibrosis : Official Journal of the European Cystic Fibrosis Society·Oscar Moran, Olga Zegarra-Moran
Jun 3, 2008·American Journal of Physiology. Lung Cellular and Molecular Physiology·Caroline NorezMarco Colombatti
Oct 27, 1999·Canadian Journal of Physiology and Pharmacology·M DuszykM W Radomski
Jun 3, 2000·American Journal of Physiology. Lung Cellular and Molecular Physiology·L J GaliettaO Zegarra-Moran
Nov 28, 2018·Molecular Diagnosis & Therapy·Giulio Cabrini

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