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 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 in normal and CF cells. Functional evaluation of CFTR function by the described method can be a useful tool to detect the expression of the CF gene transferred by adenoviral vectors for use in gene therapy trials.
Renier
M
M
Laboratory of Biochemistry, Cystic Fibrosis Center, Verona, Italy.
Tamanini
A
A
Nicolis
E
E
Rolfini
R
R
Imler
J L
JL
Pavirani
A
A
Cabrini
G
G
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D005456:Fluorescent Dyes; D018014:Gene Transfer Techniques; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D008175:Lung Neoplasms / Q000737:chemistry / Q000378:metabolism / Q000473:pathology; D015674:Mammary Neoplasms, Animal / Q000737:chemistry / Q000378:metabolism / Q000473:pathology; D008564:Membrane Potentials; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D009154:Mutation; D009298:Nasal Polyps / Q000737:chemistry / Q000378:metabolism / Q000473:pathology; D013997:Time Factors; D014407:Tumor Cells, Cultured
C505032:CFTR protein, human; D005456:Fluorescent Dyes; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D017868:Cyclic AMP-Dependent Protein Kinases