A method for the rapid detection of recombinant CFTR during gene therapy in cystic fibrosis.
We developed an assay to detect wild-type CFTR in respiratory epithelial cells with the objective to evaluate the efficacy of DNA delivery during in vivo gene transfer. The method is based on the previous observation that the common delta F508-CFTR mutant does not reach the apical membrane as does the transgene product. We thus used a monoclonal antibody, MATG 1031, raised against the first extracellular loop sequence of the CFTR protein and an immunodetection protocol lacking premature fixation or permeabilization. Specificity of MATG 1031 for its epitope was controlled by immunoblotting. In HT29-19A, 184, CAPAN-1 human cell lines, and in respiratory primary cultures, staining with MATG 1031, examined by confocal scanning laser microscopy, appeared as small dots restricted to the apical surface. No such staining was observed in NIH-3T3 fibroblasts, in the cystic fibrosis cell line CFPAC-1 or in primary cultures from cystic fibrosis patients. Apical immunostaining with MATG 1031 was restored in CFPAC-1 cells cultured at a low temperature (30 degrees C) and in CFPAC-1 cells transfected with wild-type CFTR Recombinant CFTR was also recognized in CF respiratory cells lipotransfected with wild-type CFTR plasmid DNA MATG 1031 immunostaining was further investigated under blinded conditions in primary cultures derived from nasal curettage. In all the cell cultures examined, our protocol allowed discrimination between non-CF and CF cells. We propose that this method is convenient to detect apical CFTR and may be used to monitor in vivo gene transfer.
Demolombe
S
S
laboratoire de Physiopathologie et de Pharmacologie Cellulaires et Moléculeires, Hôpital G & R Laënnec, Nautes, France.
Baró
I
I
Bebok
Z
Z
Clancy
J P
JP
Sorscher
E J
EJ
Thomas-Soumarmon
A
A
Pavirani
A
A
Escande
D
D
D016475:3T3 Cells; D000818:Animals; D002478:Cells, Cultured; D003475:Curettage / Q000379:methods; D003550:Cystic Fibrosis / Q000628:therapy*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000032:analysis* / Q000235:genetics; D015316:Genetic Therapy; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D051379:Mice; D009297:Nasal Mucosa / Q000737:chemistry* / Q000166:cytology; D011994:Recombinant Proteins / Q000032:analysis
C505032:CFTR protein, human; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator