Cystic Fibrosis Pig Model

General Characteristics

(Wild-Type Domestic Pigs)

Lifespan: 10+ years

Gestation time: 114 days

Litter Size: 8-12 piglets

Age to sexual maturity: 6 months

Adult weight: 100-180 kg

Rat

The Cystic Fibrosis Porcine Model

Three cystic fibrosis porcine models are currently available in the U.S., along with one in Europe. The first model, published in 2008, was a gene knockout (CFTR-/-), in which the endogenous CFTR is disrupted and no functional CFTR protein is produced.1,2 A second model featured a knock-in of the common human mutation, F508del (CFTRF508del/F508del), and was published shortly after.1,3 Also available is a CFTR knockout with intestinal-specific expression of a wild-type CFTR transgene (CFTR-/-;Tg FABP>pCFTR), which is sometimes referred to as the “gut-corrected” CF pig.4 In Europe, a CFTR-/- model was also developed by introducing a STOP box that terminates transcription and translation in exon 1.5 Since the development of the porcine models, CF researchers have been able to address long-standing fundamental questions about disease mechanisms, identify new aspects of the disease that had been previously unknown, and test therapies in a model that is closer to humans in size, metabolism, and CF phenotype.

Production of CF Pigs

Breeding

In the U.S., all existing CF porcine models are in a domestic pig breed, which is a Yorkshire/large white cross. The initial founder animals for the CFTR-null and CFTR-F508del herds were produced as heterozygotes via gene targeting and somatic cell nuclear transfer (SCNT).1 Heterozygote boars were then crossed with wild-type females, and a breeding herd was established. As opposed to many mouse strains, the porcine CF models are not inbred. Instead, outcrossing with traceability to a progenitor boar is utilized. Breeding male and female heterozygotes yields the expected Mendelian ratio (1:2:1). Because of the severity of the CF phenotype, homozygotes are unable to breed.

Cloning

The CF porcine models are sometimes produced by SCNT or cloning.6 In this case, a fetal fibroblast with a CFTR-/- or CFTRF508del/F508del genotype is used as a nuclear donor. Each piglet in the resulting litter is, therefore, a homozygote for the mutation. The obvious advantage of producing pigs by SCNT is that more homozygotes can be produced in fewer litters with larger study cohorts of similar ages being possible. Furthermore, each pig is genetically identical, allowing for unique experimental study designs. However, cloning for production also means the lack of littermate controls and the presence of occasional defects caused by the cloning process. The CFTR-/-;Tg FABP>pCFTR pig is produced exclusively by SCNT.

Regulatory Considerations

The United States Food and Drug Administration regulates genetically altered livestock animals under the Center for Veterinary Medicine’s Guidance for Industry #187: Regulation of Intentionally Altered Genomic DNA in Animals.7 All CF porcine models fall under this regulation; therefore, investigators must ensure compliance with all requirements when using these animals.

Maintenance

Meconium ileus in CF pigs (CFTR-/- and CFTRF508del/F508del) is nearly 100% penetrant.2,3,5 Unlike the mouse models, the intestinal obstruction cannot be overcome with diet or laxatives. Surgical correction (ostomy) is the only solution in these pigs, however, accompanying post-operative care and pancreatic enzyme supplement management is challenging. Because of this, most investigators choose to forego surgical correction and instead study the pigs during the first day or two of life.

The CFTR-/-;Tg FABP>pCFTR pig, which has a wild-type CFTR transgene driven by an intestinal-specific promoter (FABP) on the CFTR-/- background, was designed to avoid meconium ileus.4 Although this model is successful at passing meconium, the challenges of managing pancreatic insufficiency and other CF phenotypes remain. Furthermore, variability in phenotype has been reported.8

Disease Manifestations

Intestinal Disease

CFTR-/- and CFTRF508del/F508del pigs present with severe meconium obstructions at birth.2,3,5 The intestinal obstruction is most often found near the ileocecal junction with atretic bowel (microcolon) being observed distal to the blockage; however, in the European CFTR-/- pig, obstructions were observed exclusively in the large intestine.5 CF patients with severe meconium ileus are treated with a hypertonic enema, however, this is ineffective in CF pigs. Surgical correction to bypass the obstruction utilizing an ostomy has been the only successful approach to overcome meconium ileus in these genotypes. A “gut-corrected” CF pig (CFTR-/-;Tg FABP>pCFTR), in which a wild-type CFTR transgene is driven by the intestinal-specific fatty acid-binding protein (iFABP) promoter has been generated.4 These pigs have ~20% normal CFTR expression, which is sufficient to alleviate the severe meconium ileus.

 

Growth

While CFTR-/- and CFTRF508del/F508del piglets show no weight differences from their wild-type littermates at birth, the CF pigs have significantly reduced weight gain over time. This is likely due to pancreatic insufficiency and associated malnutrition, the initial intestinal phenotype, as well as lower levels of insulin-like growth factor 1 (IGF-1).9 The same is true for the CFTR-/-;Tg FABP>pCFTR pigs.

 

Pulmonary Disease

The main reasons for developing a CF porcine model were 1) the existing mouse models failed to develop spontaneous lung disease that recapitulates what is seen in patients, and 2) porcine airways (and other organs/systems) are more similar to humans in size, anatomy, and function. Indeed, all CF pigs develop airway and nasal sinus disease within weeks to months of birth.2-4,10,11 This includes infection and inflammation with a defect in innate immunity in the CF pigs causing persistent presence of bacteria. CF pig airways have structural abnormalities that had not been previously noted, such as tracheal ring irregularities and narrowed proximal airways, which contribute to obstruction.5,12 The larger size of the porcine lung has allowed for the use of human imaging tools to identify and monitor phenotypic changes, including airway wall thickening, atelectasis, and obstruction.13 This human-like size is also favorable for sampling bronchoalveolar lavage fluid and obtaining biopsy samples.

 

Hepatic Disease

CFTR-/- and CFTRF508del/F508del pigs develop liver disease similar to that seen in CF patients.14 Histology shows the presence of chronic cellular inflammation in the portal space with a focal distribution. The liver phenotype progresses with age with the appearance of fibrosis and steatosis. CF piglets also present with a mucus- and bile-filled micro gallbladder. Because wild-type CFTR expression is primarily restricted to the intestines of the CFTR-/-;Tg FABP>pCFTR pig, that model also develops human-like liver disease.

 

Pancreatic Disease

CFTR-/-, CFTRF508del/F508del, and CFTR-/-;Tg FABP>pCFTR piglets are all born with moderate to severe pancreatic disease with acinar cell destruction, duct dilation, obstruction leading to zymogen secretions, and inflammation with lymphocytes, neutrophils, and macrophages present.2,3,5,15 Pancreatic destruction progresses with age with the eventual loss of exocrine pancreatic tissue and replacement by adipose and fibrotic tissue. Because of this, the CF piglets are pancreatic insufficient and must receive enzyme supplementation in order to digest food. The CFTR-/-;Tg FABP>pCFTR piglets have a similar pancreatic phenotype.4

 

Reproductive Dysfunction

Male: CFTR-/-, CFTRF508del/F508del, and CFTR-/-;Tg FABP>pCFTR male piglets all present with reproductive tract abnormalities, including atretic vas deferens and epididymis, which ranges from partial to complete.2-5,16 Even if CF pigs reached breeding age, they would likely be infertile. Heterozygotes can breed normally.

Female: Female fertility has not been extensively studied in CF pigs; however, they would ultimately be unexpected to live to reproductive maturity. Heterozygotes can breed normally.

Available Strains

CF pig line Mutation Reference Availability
CFTR-/- (U.S.) Exon 10 disruption [1] A
CFTRF508del/F508del Knock-in of F508del mutation [1] A
CFTR-/-;Tg FABP>pCFTR CFTR-/- w/WT CFTR under iFABP promoter [1] A
CFTR-/- (European) Disruption of start codon in Exon 1 [5]

A = Exemplar Genetics (www.exemplargenetics.com)

Contact for Information or Questions about this Model

Exemplar Genetics is the exclusive commercial distributor of the CF pigs in the U.S. To learn more about these models, please contact aaron.boyd@exemplargenetics.com or contact@exemplargenetics.com or visitwww.exemplargenetics.com.

References and Reviews

1. Rogers CS, Hao Y, Rokhlina T, Samuel M, Stoltz DA, Li Y, Petroff E, Vermeer DW, Kabel AC, Yan Z, Spate L, Wax D, Murphy CN, Rieke A, Whitworth K, Linville ML, Korte SW, Engelhardt JF, Welsh MJ, Prather RS. Production of CFTR-null and CFTR-DeltaF508 heterozygous pigs by adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer. J Clin Invest. 2008;118(4):1571-7. Epub 2008/03/08.
2. Rogers CS, Stoltz DA, Meyerholz DK, Ostedgaard LS, Rokhlina T, Taft PJ, Rogan MP, Pezzulo AA, Karp PH, Itani OA, Kabel AC, Wohlford-Lenane CL, Davis GJ, Hanfland RA, Smith TL, Samuel M, Wax D, Murphy CN, Rieke A, Whitworth K, Uc A, Starner TD, Brogden KA, Shilyansky J, McCray PB, Jr., Zabner J, Prather RS, Welsh MJ. Disruption of the CFTR gene produces a model of cystic fibrosis in newborn pigs. Science. 2008;321(5897):1837-41.
3. Ostedgaard LS, Meyerholz DK, Chen JH, Pezzulo AA, Karp PH, Rokhlina T, Ernst SE, Hanfland RA, Reznikov LR, Ludwig PS, Rogan MP, Davis GJ, Dohrn CL, Wohlford-Lenane C, Taft PJ, Rector MV, Hornick E, Nassar BS, Samuel M, Zhang Y, Richter SS, Uc A, Shilyansky J, Prather RS, McCray PB, Jr., Zabner J, Welsh MJ, Stoltz DA. The DeltaF508 mutation causes CFTR misprocessing and cystic fibrosis-like disease in pigs. Science translational medicine. 2011;3(74):74ra24.
4. Stoltz DA, Rokhlina T, Ernst SE, Pezzulo AA, Ostedgaard LS, Karp PH, Samuel MS, Reznikov LR, Rector MV, Gansemer ND, Bouzek DC, Alaiwa MH, Hoegger MJ, Ludwig PS, Taft PJ, Wallen TJ, Wohlford-Lenane C, McMenimen JD, Chen JH, Bogan KL, Adam RJ, Hornick EE, Nelson GAt, Hoffman EA, Chang EH, Zabner J, McCray PB, Jr., Prather RS, Meyerholz DK, Welsh MJ. Intestinal CFTR expression alleviates meconium ileus in cystic fibrosis pigs. J Clin Invest. 2013;123(6):2685-93.
5. Klymiuk N, Mundhenk L, Kraehe K, Wuensch A, Plog S, Emrich D, Langenmayer MC, Stehr M, Holzinger A, Kröner C, Richter A, Kessler B, Kurome M, Eddicks M, Nagashima H, Heinritzi K, Gruber AD, Wolf E. Sequential targeting of CFTR by BAC vectors generates a novel pig model of cystic fibrosis. J Mol Med (Berl). 2012 May;90(5):597-608. doi: 10.1007/s00109-011-0839-y.
6. Walker SC, Shin T, Zaunbrecher GM, Romano JE, Johnson GA, Bazer FW, Piedrahita JA. A highly efficient method for porcine cloning by nuclear transfer using in vitro-matured oocytes. Cloning Stem Cells. 2002;4(2):105-12.
7. FDA. Guidance for Industry #187: Regulation of Genetically Engineered Animals Containing Heritable Recombinant DNA Constructs 2009. Available from the FDA
8. Ballard ST, Evans JW, Drag HS, Schuler M. Pathophysiologic Evaluation of the Transgenic Cftr "Gut-Corrected" Porcine Model of Cystic Fibrosis. Am J Physiol Lung Cell Mol Physiol. 2016 Oct; 311(4):L779-787.
9. Rogan MP, Reznikov LR, Pezzulo AA, Gansemer ND, Samuel M, Prather RS, Zabner J, Fredericks DC, McCray PB, Jr., Welsh MJ, Stoltz DA. Pigs and humans with cystic fibrosis have reduced insulin-like growth factor 1 (IGF1) levels at birth. Proc Natl Acad Sci U S A. 2010;107(47):20571-5.
10. Stoltz DA, Meyerholz DK, Pezzulo AA, Ramachandran S, Rogan MP, Davis GJ, Hanfland RA, Wohlford-Lenane C, Dohrn CL, Bartlett JA, Nelson GAt, Chang EH, Taft PJ, Ludwig PS, Estin M, Hornick EE, Launspach JL, Samuel M, Rokhlina T, Karp PH, Ostedgaard LS, Uc A, Starner TD, Horswill AR, Brogden KA, Prather RS, Richter SS, Shilyansky J, McCray PB, Jr., Zabner J, Welsh MJ. Cystic fibrosis pigs develop lung disease and exhibit defective bacterial eradication at birth. Science translational medicine. 2010;2(29):29ra31.
11. Stoltz DA, Meyerholz DK, Welsh MJ. Origins of cystic fibrosis lung disease. N Engl J Med. 2015;372(4):351-62.
12. Meyerholz DK, Stoltz DA, Namati E, Ramachandran S, Pezzulo AA, Smith AR, Rector MV, Suter MJ, Kao S, McLennan G, Tearney GJ, Zabner J, McCray PB, Jr., Welsh MJ. Loss of cystic fibrosis transmembrane conductance regulator function produces abnormalities in tracheal development in neonatal pigs and young children. Am J Respir Crit Care Med. 2010;182(10):1251-61.
13. Adam RJ, Abou Alaiwa MH, Bouzek DC, Cook DP, Gansemer ND, Taft PJ, Powers LS, Stroik MR, Hoegger MJ, McMenimen JD, Hoffman EA, Zabner J, Welsh MJ, Meyerholz DK, Stoltz DA. Postnatal airway growth in cystic fibrosis piglets. J Appl Physiol (1985). 2017;123(3):526-33.
14. Meyerholz DK, Stoltz DA, Pezzulo AA, Welsh MJ. Pathology of gastrointestinal organs in a porcine model of cystic fibrosis. Am J Pathol. 2010;176(3):1377-89.
15. Gibson-Corley KN, Meyerholz DK, Engelhardt JF. Pancreatic pathophysiology in cystic fibrosis. J Pathol. 2016;238(2):311-20.
16. Pierucci-Alves F, Akoyev V, Stewart JC, 3rd, Wang LH, Janardhan KS, Schultz BD. Swine models of cystic fibrosis reveal male reproductive tract phenotype at birth. Biol Reprod. 2011;85(3):442-51.