Assessment of adenoviral constructs and CFTR functional restoration using low temperature rescue.
(A) Primary AEC from children with CF were seeded into 96-well plates, grown to confluence and transduced with eYFP alone or in combination with positive controls STX-8 or BCAP-31 shRNA for 96 hours prior to performing the reporter assay. Knock-down of STX8 or BCAP-31 resulted in functional CFTR indicated by quenching of eYFP fluorescence signals. (B) Following transduction of pAECCF with eYFP and p.Phe508del cells were returned to 37°C incubator for 72 hours, followed by incubation at 27°C for 24 hours prior to halide assay. Halide assay demonstrated activation of the CFTR channels resulting in quenching of the eYFP fluorescence signal. This indicates the feasibility of using halide assay as a tool to measure functional restoration of CFTR channel.