Class
Article
College
College of Science
Presentation Type
Poster Presentation
Abstract
Cystic Fibrosis (CF) is a recessive human genetic disease that is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. This gene is responsible to transport Cl- and HCO3- anions in epithelial cells. Previously, we generated CFTR-/- lambs using CRISPR/Cas9 and SCNT techniques. The CFTR-/- lambs display many features similar to human CF disease, including meconium ileus (MI), pancreatic fibrosis, portal fibrosis and biliary hyperplasia, small gallbladder, and absence of vas deferens. In CF patients, MI affects only 15-20% of human babies, whereas it was observed in 100% of newborn CFTR-/- lambs and was the primary cause of death. We here hypothesized that the transgenic expression of the ovine CFTR cDNA under regulation of an intestinal-specific expression promoter would promote the correction of MI in CFTR-/- sheep. In this study, we are constructing three potential vectors with different promoters to be evaluated prior to the generation of transgenic animals. Rat intestinal Fatty Acid Binding Protein (iFABP), rat liver Fatty Acid Binding Protein (LFABP), and Villin1 promoters have already been characterized and successfully used for intestinal-specific expression. After digestion and ligation cloning, the three constructs will be sequenced to confirm the presence of all segments (promoter, cDNA, and vector) in the correct orientation. Subsequently, we plan to evaluate the transient gene expression of the constructs in CaCo-2 cells to ensure they are fully functional. Therefore, we will construct the pcDNA3.1>promoter>CFTR expression vector in order to generate intestine-CFTR transgenic CFTR-/- sheep.
Start Date
4-9-2020 11:00 AM
End Date
4-9-2020 12:00 PM
Construction of Candidate Vectors for Correction of the Intestinal CFTR Gene Expression in Cystic Fibrosis Sheep Fetal Fibroblast Cells
Cystic Fibrosis (CF) is a recessive human genetic disease that is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. This gene is responsible to transport Cl- and HCO3- anions in epithelial cells. Previously, we generated CFTR-/- lambs using CRISPR/Cas9 and SCNT techniques. The CFTR-/- lambs display many features similar to human CF disease, including meconium ileus (MI), pancreatic fibrosis, portal fibrosis and biliary hyperplasia, small gallbladder, and absence of vas deferens. In CF patients, MI affects only 15-20% of human babies, whereas it was observed in 100% of newborn CFTR-/- lambs and was the primary cause of death. We here hypothesized that the transgenic expression of the ovine CFTR cDNA under regulation of an intestinal-specific expression promoter would promote the correction of MI in CFTR-/- sheep. In this study, we are constructing three potential vectors with different promoters to be evaluated prior to the generation of transgenic animals. Rat intestinal Fatty Acid Binding Protein (iFABP), rat liver Fatty Acid Binding Protein (LFABP), and Villin1 promoters have already been characterized and successfully used for intestinal-specific expression. After digestion and ligation cloning, the three constructs will be sequenced to confirm the presence of all segments (promoter, cDNA, and vector) in the correct orientation. Subsequently, we plan to evaluate the transient gene expression of the constructs in CaCo-2 cells to ensure they are fully functional. Therefore, we will construct the pcDNA3.1>promoter>CFTR expression vector in order to generate intestine-CFTR transgenic CFTR-/- sheep.