Experiments were performed with CF (CFBE41o−) [51 (link)] and normal (16HBE14o−) [20 (link)] human bronchial epithelial cell lines. The CFBE41o− cell line was originally derived from a bronchial tissue isolate of a CF patient homozygous for the ΔF508 CFTR mutation and immortalized with the pSVori− plasmid that contained a replication-deficient simian virus 40 (SV40) genome [22 (link),25 (link),52 (link),53 (link)]. For the generation of CF cells complemented with wtCFTR and ΔF508CFTR, the parental CFBE41o− cell line was transfected by electroporation (nucleofection; Amaxa Biosystems, Germany) with an Epstein-Barr virus (EBV)-based episomal expression vector, pCEP4β (InVitrogen, Carlsbad, CA) containing either the 6.2 kb full-length wtCFTR cDNA (derived from pBQ6.2, a gift from L-C Tsui and J Rommens) [33 (link)] or the 4.7 kb ΔF508CFTR cDNA, respectively. The 4.7 kb ΔF508CFTR cDNA contained a TTT deletion at the ΔF508 locus rather than the naturally occurring CTT [54 (link),55 (link)] thereby making it possible to differentiate between the expression of endogenous ΔF508CFTR and the plasmid derived ΔF508CFTR. Transfected CFBE41o− cells were grown in the presence of 200–500 µg/ml hygromycin B to select for clones of cells that contained the transfected plasmid. Resistant clones were isolated, expanded and characterized. PCR, reverse transcriptase PCR (RT-PCR), and quantitative PCR and RT-PCR (Q-PCR and QRT-PCR, respectively) were used to confirm the presence and amount of the CFTR transgene and its expression, respectively. Several stable clones were identified and two clones expressing the 6.2 kb wtCFTR cDNA (CFBE41o− c7-6.2wt and CFBE41o− c10-6.2wt) and one expressing the 4.7 kb ΔF508CFTR cDNA (CFBE41o− c4-4.7ΔF) were characterized further. The clones were selected based on their level of transgene derived CFTR mRNA expression. The 16HBE14o− cell line was used as a reference for the expression of endogenous wtCFTR that results in cAMP-dependent Cl transport observed in the normal airway epithelium. Cells were grown in flasks coated with an extracellular matrix cocktail comprised of human fibronectin (BD Biosciences), Vitrogen (Cohesion, Inc.), and bovine serum albumin (Biosource/Biofluids) [12 (link),56 ] in MEM cell culture medium supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate under 5% CO2 at 37°C.