Regulated Expression of the Human CFTR Gene in Epithelial Cells

Abstract

We developed an epithelium-specific, inducible cystic fibrosis transmembrane conductance regulator (CFTR) expression system. In this system we used a human cytokeratin 18 expression cassette to drive epithelium-specific expression of the reverse tetracycline transactivator (rtTA), which turns on CFTR expression from a Tet-inducible promoter in the presence of doxycycline. CFTR expression was monitored by reverse-transcription polymerase chain reaction, immunostaining, and Western blotting. We confirmed that protein expression was dose-dependent in double stable transfected cell lines, with no detectable protein in the absence of doxycycline. However, low levels of CFTR mRNA could be detected in the uninduced state. When clones capable of inducing high levels of CFTR expression were analyzed, we observed a decrease in cell proliferation, consistent with reports in other cell lines (NIH3T3 and BTS). We generated transgenic mice expressing rtTA from the K18 expression cassette and demonstrated that the system retained its tissue specificity for lacZ reporter expression in vivo. When mice were induced with doxycycline, high levels of expression were found in the trachea, upper bronchi, and submucosal glands. Therefore, this inducible system can improve our understanding of the role of CFTR in the lung and should help in the design of safe and effective CF therapies.

Nonsteroidal anti-inflammatory drugs upregulate function of wild-type and mutant CFTR

“…By generating a doxycycline-controlled expression system in IB3-1 cells, a new cell line, named IB3-8-3-7, which expresses wild-type CFTR [6], was created. IB3-1 cells and IB3-8-3-7 cells were cultured in LHC-8 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 5% foetal bovine serum (Invitrogen) and 150 mg?mL Heterologous expression of c-aminobutyric acid receptors IB3-8-3-7 cells were transiently transfected with cDNAs encoding human A-type, c-aminobutyric acid (GABA) receptors (GABA A R)-a1, -b2, and -c2L subunit isoforms (1:1:1 ratio) by using Lipofectamine 2000 (Invitrogen).…”

“…The doxycycline-induced expression of wild-type CFTR in IB3-8-3-7 cells [6] was determined by immunocytochemistry ( fig. 1).…”

Relationships among CFTR expression, HCO 3 − secretion, and host defense may inform gene- and cell-based cystic fibrosis therapies

“…Basolateral CFTR might be less of a limitation for HCO 3 − than for Cl − secretion; the transepithelial electrochemical gradients for HCO 3 − suggest that basolateral CFTR expression might not have adverse effects on HCO 3 − secretion or ASL pH under equilibrium conditions. Another consideration is that overexpressing CFTR has been reported to inhibit cell proliferation, arrest cell growth, increase cell volume, depolarize the cell membrane, and alter CFTR function (61)(62)(63).…”

Gene Therapy Progress and Prospects: Cystic fibrosis

“…To this end, epithelium-specific regulated cassettes are being developed. 62 Each of the vectors and technologies will have to be tested in relevant in vivo models. Ideally, in contrast to CF mice, these would reproduce the lung pathology seen in CF patients.…”