205 Towards the correction of meconium ileus with cystic fibrosis transmembrane conductance regulator (CFTR) intestinal expression in CFTR knockout sheep

Abstract

Cystic fibrosis (CF) is a human autosomal genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is responsible for Cl − anion transport in epithelial cells. We previously generated CFTR +/− and CFTR −/− lambs using CRISPR/Cas9 and somatic cell nuclear transfer (SCNT) techniques. The CFTR −/− lambs display many features similar to humans with CF, including meconium ileus (MI), pancreatic fibrosis, portal fibrosis and biliary hyperplasia, small gallbladder, and absence of the vas deferens. Although MI affects only 15 to 20% of human babies with CF, it was observed in 100% of newborn CFTR −/− lambs and was the primary cause of death. We here hypothesized that the transgenic expression of ovine CFTR cDNA under regulation of the rat intestinal fatty acid binding protein (iFABP) promoter would promote the correction of MI in CFTR −/− sheep. In this study, we constructed the pcDNA3.1>iFABP>CFTR expression vector in order to generate iFABP>CFTR transgenic CFTR −/− sheep. PCR and sequence analysis of all junction regions confirmed the presence of the inserts in the vector. We transfected male and female CFTR −/− sheep fetal fibroblasts with the construct. Three days after transfection, limiting dilution and G418 selection were used for the isolation of resistant single cell derived colonies. A total of 15 male colonies containing the pcDNA3.1>iFABP>CFTR vector was confirmed by PCR and sequence analysis. Three male cell colonies were used as nuclear donors for SCNT. In total, 129 one-cell-stage cloned embryos were generated and transferred into 11 estrus-synchronized recipients. Four recipients (4/11; 36.3%) were confirmed pregnant at Day 40 to 45 of gestation. One pregnancy went to term and resulted in the birth of a single lamb. The necropsy indicated the same abnormalities, including MI, seen in CFTR −/− lambs. A PCR assay confirmed the presence of the iFABP>CFTR transgene in the genome of the cloned offspring. The lack of MI correction is likely due to the low expression of CFTR in this particular colony. Western blot analysis is in progress. Additional embryo transfers will be performed using different single-cell-derived colonies.