Production of CFTR-null and CFTR-DeltaF508 heterozygous pigs by adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer

Abstract

Progress toward understanding the pathogenesis of cystic fibrosis (CF) and developing effective therapies has been hampered by lack of a relevant animal model. CF mice fail to develop the lung and pancreatic disease that cause most of the morbidity and mortality in patients with CF. Pigs may be better animals than mice in which to model human genetic diseases because their anatomy, biochemistry, physiology, size, and genetics are more similar to those of humans. However, to date, gene-targeted mammalian models of human genetic disease have not been reported for any species other than mice. Here we describe the first steps toward the generation of a pig model of CF. We used recombinant adeno-associated virus (rAAV) vectors to deliver genetic constructs targeting the CF transmembrane conductance receptor (CFTR) gene to pig fetal fibroblasts. We generated cells with the CFTR gene either disrupted or containing the most common CF-associated mutation (DeltaF508). These cells were used as nuclear donors for somatic cell nuclear transfer to porcine oocytes. We thereby generated heterozygote male piglets with each mutation. These pigs should be of value in producing new models of CF. In addition, because gene-modified mice often fail to replicate human diseases, this approach could be used to generate models of other human genetic diseases in species other than mice.

Figure 1. CFTR expression in pig fetal fibroblasts.

Data are quantitative RT-PCR of pig CFTR mRNA relative to GAPDH in primary pig fetal fibroblasts, nasal epithelia, and rectal epithelia. Similar results were obtained on 2 other occasions.

Figure 2. Schematic of targeting constructs for homologous recombination for CFTR-null and CFTR-ΔF508.

Exons 8–11 of pig CFTR are depicted as black boxes. Neo^R contains a neomycin resistance cDNA (orange) driven by the phosphoglycerate kinase (PGK) promoter (yellow) and flanked by loxP sites (blue). The engineered stop codon is indicated in the CFTR-null targeting vector. The inverted terminal repeats (ITRs) are at both ends of the targeting construct. Position of probes for Neo^R and CFTR Southern blots are indicated. PCR screen primers are depicted as arrowheads. BglII sites are indicated. Not drawn to scale.

Figure 3. Photo of the first CFTR^+/– piglet taken at 1 day of age.

Figure 4. Southern blot of genomic DNA from CFTR-targeted pigs.

BglII-digested genomic DNA was hybridized with a probe that detects pig CFTR downstream of the targeting vector boundary, shown in Figure 2. CFTR-null and CFTR-ΔF508–targeted alleles produced an approximately 9.7-kb band, and the wild-type band is approximately 7.9 kb. (A) CFTR-null. Lanes 1–11 contain DNA from individual cloned pigs. Note that pig 10 was wild type. WT well contains DNA from a wild-type control. (B) CFTR-ΔF508. Lanes 1–5 contain DNA from individual cloned pigs. Note that pig 4 was wild type. WT well contains DNA from a wild-type control.

Figure 5. CFTR mRNA expression in CFTR^+/– and CFTR⁺/ΔF508 pigs.

(A) Quantitative RT-PCR was used to measure wild-type CFTR mRNA levels in rectal epithelial samples from CFTR^+/– and wild-type pigs. (B) Quantitative RT-PCR was used to measure ΔF508-CFTR mRNA relative to wild-type mRNA levels in CFTR⁺/ΔF508 and wild-type pigs. Error bars represent SD.