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Science 321 (5897): 1837-1841

Copyright © 2008 by the American Association for the Advancement of Science

Disruption of the CFTR Gene Produces a Model of Cystic Fibrosis in Newborn Pigs

Christopher S. Rogers1*, David A. Stoltz1*, David K. Meyerholz2*, Lynda S. Ostedgaard1, Tatiana Rokhlina1, Peter J. Taft1, Mark P. Rogan1, Alejandro A. Pezzulo1, Philip H. Karp1,3, Omar A. Itani1, Amanda C. Kabel1, Christine L. Wohlford-Lenane4, Greg J. Davis1, Robert A. Hanfland5, Tony L. Smith5, Melissa Samuel6, David Wax6, Clifton N. Murphy6, August Rieke6, Kristin Whitworth6, Aliye Uc4, Timothy D. Starner4, Kim A. Brogden7, Joel Shilyansky5, Paul B. McCray, Jr.4, Joseph Zabner1, Randall S. Prather6, and Michael J. Welsh1,3,8{dagger}

1 Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
2 Department of Pathology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
3 Howard Hughes Medical Institute (HHMI), Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
4 Department of Pediatrics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
5 Department of Surgery, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
6 Division of Animal Sciences, University of Missouri, Columbia, MO 65211, USA.
7 Department of Periodontics and Dows Institute for Dental Research, College of Dentistry, University of Iowa, Iowa City, IA 52242, USA.
8 Department of Molecular Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.

Figure 1 Fig. 1.. CFTR/ piglets appear normal at birth. (A) Upper panel depicts insertion into porcine CFTR exon 10 of a phosphoglycerate kinase (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) is indicated. The second and third panels show genotyping by PCR and Southern blot analysis of genomic DNA. Lanes C1, C2, and C3 contain controls of CFTR+/+, +/, and / DNA, respectively. The fourth panel shows Northern blot analysis 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 piglets, respectively). The bottom panel shows immunoprecipitation (IP) and phosphorylation of CFTR plus recombinant CFTR in baby hamster kidney cells. (B) First litter containing piglets of all three genotypes. (C) Birth weights. Mean ± SD of weights: 1.31 ± 0.24 kg for CFTR+/+ (orange), 1.35 ± 0.28 kg for CFTR+/ (green), and 1.31 ± 0.23 kg for CFTR/ (blue) animals. (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 (4',6'-diamidino-2-phenylindole, blue). See also fig. S1. Scale bars, 10 µm. (E) Tracings of in vivo nasal Vt measured in newborn pigs. 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 (E). Data from four CFTR+/+ and four CFTR+/ piglets (gray squares) were not statistically different and were combined and compared with data from five CFTR/ piglets (blue circles). 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. [View Larger Version of this Image (86K GIF file)]

Figure 2 Fig. 2.. CFTR/ piglets develop meconium ileus. (A) Schematic shows some clinical and histopathological CF manifestations. Pathological abnormalities are present before clinical disease becomes apparent. (B) Weight after birth. Animals were fed colostrum and milk-replacer. n = 7 CFTR+/+ and 4 CFTR/ piglets. Data are mean ± SEM. *P < 0.05. (C) Gross appearance of gastrointestinal tract. Piglets were fed colostrum and milk-replacer for 30 to 40 hours and then euthanized. Stomach, black asterisk; small intestine, arrowheads; pancreas, white arrow; rectum, white asterisk; and spiral colon, black arrow. Of 16 CFTR/ piglets, the obstruction occurred in the small intestine in 7 animals and in spiral colon in 9. (D and E) Microscopic appearance of the ileum (D) and colon (E). Hematoxylin and eosin (H&E) stain. Scale bars, 1 mm. Images are representative of severe meconium ileus occurring in 16 out of 16 (16/16) CFTR/ piglets. [View Larger Version of this Image (72K GIF file)]

Figure 3 Fig. 3.. CFTR/ piglets have exocrine pancreatic destruction and liver and gallbladder abnormalities. (A) Gross appearance of pancreas. Scale bar, 0.5 cm. (B) Loss of parenchyma in the CFTR/ pancreas. H&E stain. Scale bars, 500 µm. (C) Pancreatic ducts and islets of Langerhans (arrowheads). Scale bars, 100 µm. (D) CFTR/ ductules and acini dilated by eosinophilic inspissated material that formed concentrically lamellar concretions (arrows and inset). H&E stain. Scale bars, 33 µm. (E) Ducts within the CFTR/ pancreas. H&E stain, left; periodic acid–Schiff (PAS) stain, right. Scale bars, 50 µm. (F) Microscopic appearance of liver. H&E stain. Arrows indicate focal expansion of portal areas by chronic cellular inflammation. Scale bars, 100 µm. (G) Gross appearance of gallbladder. When the CFTR+/+ gallbladder was sectioned, bile drained away rapidly with collapse of the mucosal wall. CFTR/ bile was congealed (arrow) and retained in the lumen of a smaller gallbladder. Scale bar, 0.5 cm. (H) Microscopic appearance of gallbladder. CFTR/ gallbladders had congealed, inspissated bile (asterisk) with variable mucus production (arrows, H&E stain) highlighted as a magenta color in PAS stained tissue. Scale bars, 500 µm. Images are representative of severe pancreatic lesions (15/15 CFTR/ piglets), mild-to-moderate liver lesions (3/15), and mild-to-severe gallbladder/duct lesions (15/15). [View Larger Version of this Image (92K GIF file)]

Figure 4 Fig. 4.. The lungs of newborn CFTR/ and CFTR+/+ piglets appear normal. (A) Microscopic appearance of lung from piglets <12 hours old. H&E staining. Scale bars, 1 mm (left) and 50 µm (right). (B) Bronchial epithelia and submucosal glands. H&E staining. Scale bars, 50 µm. Images are representative of the lack of lesions in 15 out of 15 CFTR/. [View Larger Version of this Image (125K GIF file)]

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