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

Generation of Sheep Models of Cystic Fibrosis by Gene Editing and Cloning

Cystic Fibrosis (CF) is a genetic disease that affects over 80,000 people worldwide and is caused by mutations that disrupt the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. This gene encodes a channel protein that regulates ion transport in cells. The most common mutation in the human CFTR gene is F508del, which is characterized by the deletion of a phenylalanine amino acid in the CFTR protein. The second most common mutation is G542X, a protein synthesis “stop” mutation that prevents the production of the CFTR channel protein. An impediment that prevents the further understanding CF is the limited ability of most animal models to recapitulate some aspects of CF observed in humans. Sheep be a relevant animal model for CF due to the similarity of CFTR gene/protein, anatomy, physiology, body weight, and size between sheep and humans. We hypothesized that a sheep models of CF disease could replicate the severe abnormalities seen in human CF patients. Our CF sheep models exhibit severe abnormalities consistent with CF pathology in human patients. The primary cause of death of CF sheep is the intestinal obstruction that occurred in all CF lambs. Thereby, we aimed to genetically modify CF sheep to create animal models to better understand initiation and progression of CF and for the discovery of new therapeutics to treat CF patients

Mapping of QTL on chromosomes 1, 2, 3, 12, 14, 15 and X in pigs : characteristics carcass and quality of meat

A realização do presente estudo teve como objetivo mapear Quantitative Trait Loci (QTL) de carcaça e qualidade de carne em uma população F2 de suínos desenvolvida pelo cruzamento de dois reprodutores da raça brasileira Piau com 18 fêmeas comerciais (Landrace x Large White x Pietrain). O mapa de ligação para essa população foi construído após a genotipagem de 684 animais para 35 marcadores microssatélites. Os dados foram analisados pelo mapeamento por intervalo usando-se sexo, lote e genótipo halotano como efeitos fixos e peso de carcaça ao abate, peso da carcaça direita e idade ao abate como covariáveis. Um total de 18 QTLs foi encontrado; os QTLs para maior espessura de toucinho na região da copa, na linha dorsolombar, e a perda por cozimento foram significativos em nível de 5% genômico. A característica espessura de toucinho foi essencialmente associada aos alelos da raça Piau, conhecido como porco tipo banha. As informações dos QTLs significativos encontrados servem para futuros estudos de mapeamento fino para identificação de genes a serem usados em conjunto com os métodos tradicionais de seleção, para melhorar a eficiência dos programas de melhoramento, assim como prover informação acerca da fisiologia envolvida no desenvolvimento das características quantitativas dos suínos.The accomplishment of the present study had as objective to map Quantitative Trait Loci (QTL) associated to carcass and quality traits in a F2 pig population developed by mating two Brazilian Piau breed sires with 18 dams from a commercial line (Landrace × Large White × Pietrain). The linkage map for this population was constructed after genotyping the 684 animals for 35 microsatellite markers. Data were analyzed by interval mapping using sex, batch and halothane genotype as fixed effects and carcass weight at slaughter, direct carcass weight and slaugher age as covariables. A total of 18 QTL were identified, the QTL for higher backfat thickness on the shoulder region and cooking loss was significant at 5% genome-wise level. The backfat thickness trait was mainly associated with the Piau breed allele, known as a fat pig. The information of the significant QTL detected in this study is useful for future fine-mapping studies for identification of genes and might be used together with traditional selection methods to improve the efficiency of breeding programs, moreover, this information can also provide new insights to the understanding of the physiology of the quantatiative traits in pigs

Mapping of QTL on chromosomes 1, 2, 3, 12, 14, 15 and X in pigs : characteristics carcass and quality of meat

A realização do presente estudo teve como objetivo mapear Quantitative Trait Loci (QTL) de carcaça e qualidade de carne em uma população F2 de suínos desenvolvida pelo cruzamento de dois reprodutores da raça brasileira Piau com 18 fêmeas comerciais (Landrace x Large White x Pietrain). O mapa de ligação para essa população foi construído após a genotipagem de 684 animais para 35 marcadores microssatélites. Os dados foram analisados pelo mapeamento por intervalo usando-se sexo, lote e genótipo halotano como efeitos fixos e peso de carcaça ao abate, peso da carcaça direita e idade ao abate como covariáveis. Um total de 18 QTLs foi encontrado; os QTLs para maior espessura de toucinho na região da copa, na linha dorsolombar, e a perda por cozimento foram significativos em nível de 5% genômico. A característica espessura de toucinho foi essencialmente associada aos alelos da raça Piau, conhecido como porco tipo banha. As informações dos QTLs significativos encontrados servem para futuros estudos de mapeamento fino para identificação de genes a serem usados em conjunto com os métodos tradicionais de seleção, para melhorar a eficiência dos programas de melhoramento, assim como prover informação acerca da fisiologia envolvida no desenvolvimento das características quantitativas dos suínos.The accomplishment of the present study had as objective to map Quantitative Trait Loci (QTL) associated to carcass and quality traits in a F2 pig population developed by mating two Brazilian Piau breed sires with 18 dams from a commercial line (Landrace × Large White × Pietrain). The linkage map for this population was constructed after genotyping the 684 animals for 35 microsatellite markers. Data were analyzed by interval mapping using sex, batch and halothane genotype as fixed effects and carcass weight at slaughter, direct carcass weight and slaugher age as covariables. A total of 18 QTL were identified, the QTL for higher backfat thickness on the shoulder region and cooking loss was significant at 5% genome-wise level. The backfat thickness trait was mainly associated with the Piau breed allele, known as a fat pig. The information of the significant QTL detected in this study is useful for future fine-mapping studies for identification of genes and might be used together with traditional selection methods to improve the efficiency of breeding programs, moreover, this information can also provide new insights to the understanding of the physiology of the quantatiative traits in pigs