Cystic Fibrosis Ferret Model

General Characteristics

Lifespan: 7-9 years

Gestation time: 42 days

Age to sexual maturity: 4-6 months

Adult weight: Females 600-1000 grams, Males 900-1400 grams

Ferret

The Cystic Fibrosis Ferret Model

The CFTR knockout ferret was created in 2008,1 and its phenotype was published in 2010.2 Since then, a G551D-CFTR model was created that reduces the difficulty of rearing and aging CF ferrets when a CFTR potentiator is given in utero and after birth.3 The CF ferret model has aided researchers in studying pathogenesis of lung, exocrine pancreatic disease, and CF-related diabetes.

General Husbandry of CF Ferrets

Generation

The first CFTR knockout ferret was generated using somatic cell nuclear transfer technology. The first-generation G551D model was created using somatic cell nuclear transfer and contains a neomycin expression cassette in a CFTR intron. This leads to hypomorphic expression of G551D-CFTR mRNA at a level of 50% that of a WT allele. Thus, the maximal recovery of G551D/G551D-CFTR function with VX-770 using in vitro epithelial models is 25% that of WT. A second-generation G551D-CFTR model (CFGM-KI) was generated using CRISPR/Cas9 and produced a “clean” mutation that is currently being characterized and appears to afford greater potentiation with VX-770 due to higher mRNA expression.

Maintenance

CFTR knockout ferrets suffer a high rate (~80%) of meconium ileus (MI) at birth,2-4 which is surgically inoperable, and kits must be euthanized within 24 hours after birth. The CF kits that that do not have MI (~20%) require pancreatic enzyme supplements, laxatives, and a special diet to prevent bowel obstruction and improve growth. In the absence of antibiotic therapy initiated at birth, CFTR knockout kits rapidly develop lung infections within weeks2 and have abnormal inflammatory responses in the lung.5 When symptomatically treated with antibiotics, CFTR knockout ferrets live longer (average of 105 +/-27 days), but still succumb to bacterial colonization of the lung.6 When aggressively treated with a three-antibiotic cocktail from birth, CFTR knockout ferrets live an average of 1,143 +/-77 days but still develop structural and mucoinflammatory lung disease.7 In the G551D-CFTR model, prenatal administration of the CFTR potentiator VX-770 mixed in EleCare and given orally to the pregnant jill starting at E28, greatly reduces the incidence of MI at birth and continued administration to newborns enhances growth and survivability.3 Newborns are administered VX-770 mixed in EleCare by gavage until 2 weeks of age when a syringe can be used to dose them orally. Homozygous G551D-CFTR ferrets treated with VX-770 do not require enzyme supplements or laxatives and can be fed a regular chow diet, while compound heterozygous G551D/KO CF ferrets become pancreatic insufficient by ~1 months of age and require pancreatic enzyme supplementation after weaning. Withdrawal of VX-770 from G551D-CFTR ferrets initiates disease in the lung and pancreas, with the most rapid onset of disease occurring when withdrawal is performed prior to three weeks of age.

Disease Manifestations

 

Intestinal Disease

About 80% of CF ferrets are born with meconium ileus (MI) and won’t survive more than a couple of days.2,3 Those that don’t have MI at birth are susceptible to intestinal blockage and require a special diet to survive, including pancreatic enzymes and laxatives during the first months of life.2 However, in the G551D model, the incidence of MI at birth is greatly reduced by treating the pregnant jill with VX-770 from day 28 of gestation until birth. Treating the newborn ferrets with VX-770 also decreases the susceptibility to intestinal blockage later in life and allows them to be reared much like wild type ferrets when the transgene is homozygosed.3 This first-generation G551D model has hypomorphic expression of G551D-CFTR mRNA at a level of 50% that of a WT allele. Thus, the maximal recovery of G551D/G551D-CFTR function with VX-770 using in vitro epithelial models is 25% that of WT. For this reason, only homozygous animals are fully protected from disease when reared on VX-770. A second-generation G551D-CFTR model (CFGM-KI) was generated using CRISPR/Cas9 and produced a “clean” mutation that is currently being characterized and appears to afford greater potentiation with VX-770 due to higher mRNA expression.

 

Pulmonary Disease

CFTR knockout ferrets develop bacterial lung infections within the first week of life, and even when treated symptomatically with antibiotics, they are susceptible to infection for the entirety of their lives.2,3,5,6 Although the most acute infections occurred early in life, those that survived past weaning demonstrated a slower, progressive pattern of infection and colonization. The bacteria found in the lung were diverse, with Staphylococcus, Streptococcus and Enterococcus being the most common. As in humans, the severity of the lung disease in ferrets can range from mild to severe and includes reduced mucociliary clearance, mucus obstruction of airways and submucosal glands, air trapping, and pneumonia. With the development of the G551D-CFTR ferrets, the timing of CF lung disease can be controlled by withdrawal of VX-770. In adult G551D-CFTR ferrets, withdrawal of VX-770 leads to lung colonization within three months, as detected by culturable bacteria in the bronchioalveolar lavage fluid.3

 

Pancreatic Disease

Newborn CFTR knockout animals have mild pancreatic histopathology and exocrine pancreatic disease similar to CF infants.2,8 In the first 1-2 months of life, CF ferrets experience progressive exocrine and endocrine loss accompanied with inflammation, glucose intolerance, and spontaneous hyperglycemia.4,9,10 Newborn and juvenile CF ferrets exhibit abnormally high glucose during glucose tolerance tests, as do 2-3 month old CF ferrets in mixed meal tolerance tests. Fasted newborn ferrets have an inhibited first phase insulin response when challenged with L-Arginine or glucose with a reduced plasma insulin/glucose ratio at early time points. As they age, the CFTR knockout ferrets experience both exocrine and endocrine pancreatic remodeling with a significant loss in islet mass, increasing fibrosis, and absent fecal EL-1 indicating pancreatic insufficiency.4,9-11 G551D/KO-CFTR ferrets exhibit a slightly delayed CF pancreatic phenotype and glucose abnormalities in the presence of VX-770 treatment.3 However, G551D/G551D-CFTR ferrets demonstrate nearly full protection from pancreatic disease and diabetes until VX-770 is withdrawn, after which they become pancreatic insufficient and develop glucose excursions.3

 

Reproductive Dysfunction

Male CFTR-knockout ferrets are born with either a segmentally absent or complete absence of the vas deferens. However, homozygous G551D ferrets treated in utero with VX-770 are born with an intact epididymis and vas deferens.3

Available Strains

CF Ferret Strain Transgene Reference
CFKO CFTR Knockout [2]
CFGM G551Dneo [3]
CFGM-KI G551D unpublished
CFDFM F508del unpublished

Contact for Information or Questions about this Model

e-mail: michael-winter@uiowa.edu

References and Reviews

1. Sun, X., et al., Adeno-associated virus-targeted disruption of the CFTR gene in cloned ferrets. J Clin Invest, 2008. 118(4): p. 1578-83.
2. Sun, X., et al., Disease phenotype of a ferret CFTR-knockout model of cystic fibrosis. J Clin Invest, 2010. 120(9): p. 3149-60.
3. Sun, X., et al., In utero and postnatal VX-770 administration rescues multiorgan disease in a ferret model of cystic fibrosis. Sci Transl Med, 2019. 11(485).
4. Sun, X., et al., Gastrointestinal pathology in juvenile and adult CFTR-knockout ferrets. Am J Pathol, 2014. 184(5): p. 1309-22.
5. Keiser, N.W., et al., Defective innate immunity and hyperinflammation in newborn cystic fibrosis transmembrane conductance regulator-knockout ferret lungs. Am J Respir Cell Mol Biol, 2015. 52(6): p. 683-94.
6. Sun, X., et al., Lung phenotype of juvenile and adult cystic fibrosis transmembrane conductance regulator-knockout ferrets. Am J Respir Cell Mol Biol, 2014. 50(3): p. 502-12.
7. Rosen, B.H., et al., Infection Is Not Required for Mucoinflammatory Lung Disease in CFTR-Knockout Ferrets. Am J Respir Crit Care Med, 2018. 197(10): p. 1308-1318.
8. Gibson-Corley, K.N., et al., Pancreatic pathophysiology in cystic fibrosis. J Pathol, 2016. 238(2): p. 311-20.
9. Olivier, A.K., et al., Abnormal endocrine pancreas function at birth in cystic fibrosis ferrets. J Clin Invest, 2012. 122(10): p. 3755-68.
10. Yi, Y., et al., A Transient Metabolic Recovery from Early Life Glucose Intolerance in Cystic Fibrosis Ferrets Occurs During Pancreatic Remodeling. Endocrinology, 2016. 157(5): p. 1852-65.
11. Rotti, P.G., et al., Pancreatic and Islet Remodeling in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Knockout Ferrets. Am J Pathol, 2018. 188(4): p. 876-890.