Sheep Models of F508del and G542X Cystic Fibrosis Mutations Show Cellular Responses to Human Therapeutics

Cystic Fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The F508del and G542X are the most common mutations found in US patients, accounting for 86.4% and 4.6% of all mutations, respectively. The F508del causes deletion of the phenylalanine residue at position 508 and is associated with impaired CFTR protein folding. The G542X is a nonsense mutation that introduces a stop codon into the mRNA, thus preventing normal CFTR protein synthesis. Here, we describe the generation of CFTRF508del/F508del and CFTRG542X/G542X lambs using CRISPR/Cas9 and somatic cell nuclear transfer (SCNT). First, we introduced either F508del or G542X mutations into sheep fetal fibroblasts that were subsequently used as nuclear donors for SCNT. The newborn CF lambs develop pathology similar to CFTR−/− sheep and CF patients. Moreover, tracheal epithelial cells from the CFTRF508del/F508del lambs responded to a human CFTR (hCFTR) potentiator and correctors, and those from CFTRG542X/G542X lambs showed modest restoration of CFTR function following inhibition of nonsense-mediated decay (NMD) and aminoglycoside antibiotic treatments. Thus, the phenotype and electrophysiology of these novel models represent an important advance for testing new CF therapeutics and gene therapy to improve the health of patients with this life-limiting disorder

Effect of oral polyamine supplementation pre-weaning on piglet growth and intestinal characteristics

A high proportion of piglets fail to adapt to the changing composition of their diet at weaning, resulting in weight loss and increased susceptibility to pathogens. Polyamines are present in sow milk and promote neonatal maturation of the gut. We hypothesised that oral spermine and spermidine supplementation before weaning would increase piglet growth and promote gastrointestinal development at weaning. In Experiment One, one pair of liveweight (LW)-matched piglets per litter from first and third lactation sows received 2 ml of a 0 (Control) or 463 nmol/ml spermine solution at 14, 16, 18, 20 and 22 days of age (n=6 piglets/treatment per parity). Villus height and crypt depth in the duodenum and jejunum were measured at weaning (day 23 postpartum). In Experiment Two, piglets suckling 18 first and 18 third lactation sows were used. Within each litter, piglets received 2 ml of either water (Control), 463 nmol/ml spermine solution or 2013 nmol/ml spermidine solution at 14, 16, 18, 22 and 24 days of age (n=54 piglets/treatment per sow parity). Piglets were weighed individually at 14, 18, 24 (weaning) and 61 days of age. In Experiment One, oral spermine supplementation resulted in a 41% increase in villus height, a 21% decrease in crypt depth and 79% decrease in the villus height : crypt depth ratio compared with control piglets (P<0.01). In Experiment Two, spermine and spermidine-supplemented piglets suckling first lactation sows grew faster (P<0.05) between days 14 and 18 postpartum than control piglets: 0.230±0.011 and 0.227±0.012 v. 0.183±0.012 kg/day, respectively. Spermine supplementation tended (P<0.1) to increase piglet LW gain from weaning to day 37 post-weaning compared with control piglets (0.373±0.009 v. 0.341±0.010 kg/day). In conclusion, spermine supplementation increased villus height at weaning, and appears to have the potential to improve the pre- and post-weaning growth of conventionally weaned piglets.W.H.E.J. van Wettere, N.-L. Willson, S.J. Pain and R.E.A. Forde