Figure 3.
The hG551D-CFTR (cystic fibrosis transmembrane conductance regulator) is expressed and functional in the airway. (A–C) Gene expression analysis of lungs from wild-type (WT), hG551D, and knockout (KO) rat using primers specific to rats (A), humans (B), and both (C) indicate human specificity of the sequence and appropriate expression. (D) Western blot indicates that the protein concentration in the lung is also similar to that of WT rats. (E) Blots were analyzed for density of CFTR bands compared with loading control (α-tubulin). (F) To confirm that hG551D-CFTR localized to the surface of the epithelium, tracheal sections from WT, hG551D, and KO rats were stained with antibodies against CFTR (green), cilia (red), and nuclei (blue). White bar = 10 μm. (G) Short-circuit current (Isc) analysis was performed on trachea, with representative tracings from each genotype. (H) Combined summary data indicate that the hG551D rat has a lower baseline current than the WT rat but responds specifically to increasing doses of VX-770. Statistical comparison between WT and hG551D rats is in blue; comparison between hG551D and KO rats is in red. (I) Δ currents 10 μM VX770 demonstrate a selective response to ivacaftor in the hG551D animal. (J) Δ currents after CFTR-Inh172 indicate different responses between genotypes. The CFTR-Inh172 response indicates that hG551D rats can be potentiated to approximately 50% of WT activity. *P < 0.05, **P < 0.01, and ***P < 0.001. n = 6/group. Amil = amiloride; Fsk = forskolin; ns = not significant.