Additional analysis of different epithelia other than the
Additional analysis of different epithelia, other than the intestine, in piglets carrying the ΔF508 mutation confirmed the presence of a residual CFTR function (about 6% of the wild-type function), as result of a small fraction of ΔF508-CFTR that escapes the ER and reaches the apical membrane . This result confirms previous in vitro data in cell lines carrying the same mutation and suggests that such a CFTR residual activity is not sufficient to prevent the different phenotypes in the pig.
An interesting anatomical feature of the pig is that the common bile duct and the pancreatic duct openings to the intestine are anatomically distinct . Therefore, researchers were able to perform a detailed analysis of the pancreatic and biliary fluid separately in CFTR-KO animals. The volume and pH of pancreatic fluid was found to be lower compared to WT, and protein content was increased. Bile volume in resting conditions was similar between CF and WT and the increase in response to the physiologic hormone secretin was observed only in WT piglets confirming the secretory defect in CF pigs .
A second CF animal model recently generated is the ferret . The ferret was chosen because, contrary to the mouse, its lung anatomy and cell biology closely resemble those of humans. Their gestation time of 42 days is close to the mice. The first ferret was obtained by insertion of a stop FF-MAS and the neomycin cassette into exon 10 of CFTR gene that generates a null genotype.
Similar to the mice and the pig, the intestinal phenotype in the CFTR-KO ferret is severe and about 75% of the kits dye within 24 h after birth. A disadvantage of the ferret, compared to the pig is that the small size of a ferret kit prevents the possibility of a surgical treatment. In addition to the development of meconium ileus, the nutritional status of newborn CF ferrets is severely compromised and reflects anatomical differences in their intestinal tract, mainly the lack of a cecum and a shorter intestinal transit. In those animals that were able to survive, the pancreas shows histological lesions as seen in CF patients but less severe compared to the pig model. While the liver histology of CFTR-KO ferrets appears normal, the serum analysis reveals increased levels of ALT and bilirubin with decreased levels of cholesterol [12,47].
The CFTR-KO ferrets were treated with UDCA and proton-pump inhibitors that were able to normalize the liver enzymes and improve their nutritional status, but the cholesterol remained low . Survival in these animals was also compromised by development of multifocal bronchopneumonia with severe lung damage, inflammation and bacterial colonization. Interestingly, a second study has shown that several inflammatory pathways and airway innate immune mechanisms are altered in the KO ferret before and immediately after birth.
New models and future directions With the advantage of novel genetic engineering approaches, new CF animal models are currently under development (i.e. rat, rabbit) . These animals are still being characterized and there are no data related to the liver phenotype. The rat CFTR-KO model has been generated using Zinc-finger endonuclease (ZFN) technology . This model has all the advantages of the mouse including the short time for breeding, contained husbandry costs and the availability of specie-specific molecular tools that for example are still missing for the ferret (i.e. antibodies, recombinant proteins). Some anatomical similarities with the humans, not present in the mouse, such as the presence of submucosal glands were attracting for lung researchers. Indeed, the first results report that the rat reproduces several features of CF human airway disease (i.e. mucus plugging, nasal and tracheal electrophysiological defects). However, similar to the mouse there was no evidence of spontaneous infections or inflammation in rat airways .