title: GFP-tagged CFTR transgene is functional in the G551D cystic fibrosis mouse colon.
abstract: Trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is central to its function, with the most common mutation, deltaF508, resulting in abnormal processing and trafficking. Therefore, there is a significant need to develop tools, which enable the trafficking of CFTR to be studied in vitro and in vivo. In previous studies it has been demonstrated that fusion of the green fluorescent protein (GFP) to the N-terminus of CFTR does lead to functional expression of CFTR chloride channels in epithelial cell lines. The aim of the present study was to examine whether it is possible to express GFP-tagged CFTR as a transgene in colonic and airway epithelial cells of cystic fibrosis (CF) mice and to correct the CF defect. Using the epithelial-specific human cytokeratin promoter K18, we generated bitransgenic mice cftr(G551D/G551) K18-GFP-CFTR(+/-), designated GFP mice. Transcripts for GFP-CFTR could be detected in bitransgenic mice by use of RT-PCR techniques. Expression of GFP-CFTR protein was detected specifically in the colonic epithelium by both direct GFP fluorescence and the use of an anti-GFP antibody. Ussing chamber studies showed that the ion transport defect in colon and airways observed in cftr(G551D/G551D) mice was partially corrected in the bitransgenic animals. Thus, K18-GFP-CFTR is functionally expressed in transgenic mice, which will be a valuable tool in studies on CFTR synthesis, processing and ion transport in native epithelial tissues.
authors.affiliation: Institute for Molecular Biosciences, The University of Queensland, St. Lucia QLD 4072, Australia.
authors.forename: D
authors.initials: D
authors.lastname: Oceandy
authors.forename: B
authors.initials: B
authors.lastname: McMorran
authors.forename: R
authors.initials: R
authors.lastname: Schreiber
authors.forename: B J
authors.initials: BJ
authors.lastname: Wainwright
authors.forename: K
authors.initials: K
authors.lastname: Kunzelmann
mesh_terms: D000818:Animals; D003106:Colon / Q000378:metabolism*; D003550:Cystic Fibrosis / Q000235:genetics / Q000378:metabolism* / Q000473:pathology / Q000628:therapy; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D015316:Genetic Therapy / Q000379:methods; D007413:Intestinal Mucosa / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D018181:Mice, Inbred CFTR; D008822:Mice, Transgenic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020545:Respiratory Mucosa / Q000378:metabolism / Q000473:pathology; D014132:Trachea / Q000378:metabolism / Q000473:pathology
chemical_list: D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator