1. Cystic Fibrosis: Using ferrets as a Disease Model

2. Ferret Models

3. Terms and Abbreviations CF = Cystic Fibrosis SCNT = Somatic Cell Nuclear Transfer MI = Meconium Ileus LFT = Liver Function Test ALT = Alanine Aminotransferase UDCA = Ursodeoxycholic Acid FABPi = Fatty acid binding promoter

4. Background CF affects: 1 in 3,500 newborns each year People of mixed European descent Caused by a defective CFTR gene Codes for epithelial chloride channel

5. Background CF affects many tissues: Lung Liver Pancreas Male Reproductive Tract Intestines Gall Bladder Sweat Gland

6. Why ferrets? 2 reasons: Their lung anatomy and cell biology are similar to humans They reproduce fast

7. Methodology Knockout gene was engineered HA-tagged cDNA of CFTR gene Used the FABPi promoter

8. Results 9 clones heterozygous for CFTR were bred Expected 1:2:1 ratio to create ferret models with CF, or kits

9. Results At 24 hours, CF kits were indestinguishable from littermates At 36 hours, they failed to thrive Euthanized at 48 hours

10. Results: MI Variable Penetrance MI affects the intestine It is a blockage Can cause perforations in intestine Frequency ranged for 50-100% in offspring of 9 heterzygous male parents Was statistically linked to the parents used in breeding (P< 0.047)

12. Results: Male RT Nearly all males with CF suffered from infertility Vas Deferens becomes absent throughout development Unilateral degeneration detected as early as 2 yrs old

13. Results: Male RT

14. Results: Pancreas All glands exhibited legions Ferrets were consistant with those in newborn humans with CF Significantly less severe than those seen on the Pig model Histopathology of gall bladder and liver indistinguishable from non CF littermates

16. Results: Lungs Those infants that escaped MI did not escape Lung complications 25% of neonatal deaths were not found out for those who passed MI Evidence found of pneumonia and aspiration

17. Results: Lungs ALT levels were elevated Bilirubin Elevated Cholesterol levels reduced Suggests potential liver disease No histo legions on liver, but elevated LFTs

18. Results: Liver Elevated LFTs is unclear Bile acid malabsorption by intestine Changes circulation and composition of bile acids Cholesterol levels low due to defective fatty acid absorption in the gut Oral doses of UDCA showed normalization of LFTs and ALT levels

20. Results: Nutrition Due to bad bile acids, lipids become hard to digest in CF patients. Due to impaired pancreas activity, HCO3- is not secreted into gut which lowers pH To counteract, oral proton pump is given to try and normalize gut pH

21. Conclusions There are many models out there Each one may be best suited to a specific symptom of CF Still doing research on ferrets for lungs Initial reasoning for using them is sound

22. References 1.) Disease phenotype of a ferret CFTR-knockout model of cystic fibrosis Published in Volume 120, Issue 9 (September 1, 2010) J Clin Invest. 2010;120(9):3149–3160. doi:10.1172/JCI43052. Copyright © 2010, American Society for Clinical InvestigationVolume 120, Issue 9 2.) Riordan JR, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science. 1989;245(4922):1066–1073. 3.) Egan ME. How useful are cystic fibrosis mouse models? Drug Discov Today Dis Models. 2009;6(2):35–41. 4.) Rogers CS, et al. Disruption of the CFTR gene produces a model of cystic fibrosis in newborn pigs. Science. 2008;321(5897):1837–1841.

23. All 3 Models

24. Thanks for Listening!