Adeno-associated virus-targeted disruption of the CFTR gene in cloned ferrets

Abstract

Somatic cell gene targeting combined with nuclear transfer cloning presents tremendous potential for the creation of new, large-animal models of human diseases. Mouse disease models often fail to reproduce human phenotypes, underscoring the need for the generation and study of alternative disease models. Mice deficient for CFTR have been poor models for cystic fibrosis (CF), lacking many aspects of human CF lung disease. In this study, we describe the production of a CFTR gene-deficient model in the domestic ferret using recombinant adeno-associated virus-mediated gene targeting in fibroblasts, followed by nuclear transfer cloning. As part of this approach, we developed a somatic cell rejuvenation protocol using serial nuclear transfer to produce live CFTR-deficient clones from senescent gene-targeted fibroblasts. We transferred 472 reconstructed embryos into 11 recipient jills and obtained 8 healthy male ferret clones heterozygous for a disruption in exon 10 of the CFTR gene. To our knowledge, this study represents the first description of genetically engineered ferrets and describes an approach that may be of substantial utility in modeling not only CF, but also other genetic diseases.

Figure 1. CFTR gene targeting in ferret fibroblasts.

(A) Genomic fragment containing exon 10 of the ferret CFTR gene (top) and the rAAV CFTR targeting vector (bottom). Arrows mark nested primers used for PCR screening of targeting events. Restriction sites and probes used for Southern blot confirmation of targeting events are also shown. (B) Southern blot analysis of fibroblasts derived from the CL-B96 CFTR gene–targeted 21-day NT cloned ferret embryo using restriction sites and probes marked in A. The originating fibroblast line used for targeting is shown in lanes 1 and 4, while the CFTR gene targeted fibroblasts are shown in lanes 2 and 3. Arrowheads to the right of blots indicate the nontargeted (filled) and gene-targeted (open) CFTR alleles.

Figure 2. Cloning of CFTR-targeted ferrets.

(A) Cloned CFTR-targeted ferrets. The top left panel shows the first clone (sable coat color) at 5 weeks of age with its albino noncloned foster sibling. The other panels show sable clones with ages indicated. (B) Southern blot analysis of ear fibroblast DNA from the 8 CFTR-targeted cloned ferrets (nos. 1–8) using AflII-digested genomic DNA and the indicated CFTR and neomycin probes. NT, nontargeted unrelated ferret DNA. Arrowheads indicate the nontargeted (filled) and gene-targeted (open) CFTR alleles. (C) Preweaning growth rate of CFTR-targeted ferret clones as compared with unrelated noncloned ferrets. Results are shown as the mean ± SEM for the indicated n in each group.