Figure 1. CFTR−/− piglets appear normal at birth.

A) Upper panel depicts insertion into porcine CFTR exon 10 of a PGK promoter (yellow) driving a neomycin resistance cDNA (orange), and an engineered stop codon. Position of probe (green), PCR primers (arrowheads) and BglII sites (B) are indicated. Second and third panels show genotyping by PCR and Southern blot of genomic DNA. Lanes C1, C2, and C3 contain controls of CFTR+/+, +/− and −/− DNA. Fourth panel shows northern blot of ileal CFTR and GAPDH mRNA. Consistent with the northern blot, quantitative RT-PCR of exon 10, the targeted site, detected <0.1% of CFTR transcripts in CFTR −/− ileum relative to CFTR+/+ (n=6 and 4). Fifth panel shows immunoprecipitation and phosphorylation of CFTR plus recombinant CFTR in BHK cells. B) First litter containing piglets of all three genotypes. C) Birth weights. Mean±SD of weights: 1.31±0.24 kg for CFTR+/+, 1.35±0.28 kg CFTR+/−,and 1.31±0.23 kg CFTR−/−. D) Immunocytochemistry of CFTR in airway epithelia (top) and ileum (bottom). Figures are differential interference contrast with staining for ZO-1 (a component of tight junctions, red), CFTR (green), and nuclei (DAPI, blue). See also Fig. S1. Bars, 10 μm. E) Tracings of in vivo nasal voltage (Vt) measured in newborn piglets. After baseline measurements, the following agents/solutions were sequentially added to the epithelial perfusate: amiloride (100 μM), Cl⁻-free solution, isoproterenol (10 μM), ATP (100 μM), and GlyH-101 (100 μM). F) Average nasal Vt measurements as indicated in panel E. Data from 4 CFTR+/+ and 4 CFTR+/− piglets were not statistically different and were combined and compared to data from 5 CFTR−/− piglets. Values of baseline nasal Vt for CFTR−/− piglets differed from the controls, as did the changes in Vt induced by adding amiloride, a Cl⁻-free solution, and GlyH-101 (all P<0.05). Data are mean±SEM.