Tissue-Specific Complementation of Intestinal Disease in a Transgenic CFTR-Knockout Ferret Model Generated by SCNT.

Abstract

Our laboratory recently generated a cystic fibrosis transmembrane conductance regulator gene (CFTR) knockout (KO) ferret model of cystic fibrosis (CF) using viral gene-targeting and somatic cell nuclear transfer (SCNT). Here we describe the phenotype of CFTR null ferrets and methods for genetic complementation of disease using tissue-specific transgenesis in CFTR-KO fibroblasts by SCNT. Neonatal CFTR-KO ferrets demonstrated many of the characteristics of human CF disease, including defective airway chloride transport, variably penetrant meconium ileus (MI), pancreatic and liver disease. 75% of all CFTR-KO kits die from meconium ileus with in 36-48 hrs after birth. Severe malabsorption by the gastrointestinal tract was the primary cause of death in the 25% of CF kits that escaped MI. Since a major reason for generating the CFTR-KO ferret model was to study the progression of lung disease in adult animals, methods to overcome the limitations imposed by the severe gut phenotype in this model were needed. To this end, we cloned a gut-corrected CFTR-KO ferret by somatic cell nuclear transfer using transgenic CFTR-KO fibroblasts expressing fCFTR under the direction of the intestine-specific fatty acid protein (FABPi) promoter. A transgenic construct containing the rat FABPi promoter driving the fCFTR cDNA and a floxed PGK promoter driven zeocin resistant gene cassette was engineered and transfected into ferret fibroblasts generated from a 28 day CFTR-KO embryo. The transfected cells were selected in zeocin and the pooled cells (i.e., non-clonal) were used for SCNT. Three clones were born, but only one survived the early post-natal period and passed meconium. The two additional clones born had to be euthanized within 36 hrs after birth due to severe MI with microcolon. The surviving clone was euthanized to determine the expression patterns of recombinant fCFTR and this clone had a grossly normal intestine lacking any signs of MI. Fibroblasts, liver, lung, and intestine were harvested from this MI-complemented founder clone for analysis of recombinant fCFTR tissue expression patterns prior to nuclear transfer recloning and expansion of the line. Results from analysis of this MI-complemented FABPi-fCFTR/CFTR-KO clone demonstrated the presence of the transgene cassette in the genomic DNA and recombinant fCFTR protein expression in the intestine but not in the lung and liver (endogenous fCFTR protein was expressed in all these organs from wild type ferrets but not CFTR-KO ferrets). In a separate set of experiments, we cloned non-transgenic CFTR-KO kits with fibroblasts derived from a CFTR-KO kit that suffered from severe MI with microcolon; all four clones born from this experiment suffered from MI with varying degrees of microcolon. Thus, the intestinal phenotype of cloned CFTR-KO kits appears to closely reflect that of the CFTR-KO founder. Cumulatively, these findings demonstrate the successful generation of a transgenic FABPi-fCFTR/CFTR-KO founder for which tissue-specific expression of recombinant fCFTR in the intestine can correct the MI phenotype. Currently, nuclear transfer is being performed to expand the FABPi-fCFTR/CFTR-KO line with and without CRE-excision of the PGK-Zeocin cassette. This first demonstration of generating a transgenic ferret proves the feasibility of promoter-directed complementation in the ferret by SCNT, and may significantly enhance the utility of the CF ferret model for research. (poster)