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

Department of Anatomy and Cell Biology, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242, USA.
Journal of Clinical Investigation (Impact Factor: 13.22). 05/2008; 118(4):1578-83. DOI: 10.1172/JCI34599
Source: PubMed


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.

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Available from: Ziyi Li, Aug 18, 2014
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    • "Several hurdles had to be overcome, but somatic cell nuclear transfer in ferrets (Li and Engelhardt 2003) and successful site-directed mutagenesis of the causative Cftr gene by adeno-associated viral transduction (Sun et al. 2008) eventually resulted in a ferret model that closely resembles the phenotype of human cystic fibrosis, as well as that of pig models (Sun et al. 2010). "
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    • "Recombinant adeno-associated viral vectors (rAAVs) are exceptionally versatile and powerful gene delivery vehicles (tools) for use in translational science and basic biology. Strategies have been developed to use rAAVs for gene augmentation, miRNA-and shRNA-mediated knockdown, endogenous miRNA antagonism, and genome editing using zinc finger nucleases or rAAV to induce homologous recombination (Egan et al., 1992; Cruz et al., 2007; Cideciyan et al., 2008; Sun et al., 2008; Brantly et al., 2009; Gorbatyuk et al., 2010; Li et al., 2011; Asuri et al., 2012; Ellis et al., 2012; Handel et al., 2012; Mueller et al., 2012; Xie et al., 2012). For the most part, AAV vectors lead to robust long-term in vivo transgene expression in both dividing and non-dividing cells in a myriad of cell types and animal models. "
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    • "Therefore, the development of (1) alternative animal models to study the progression of CF disease and (2) cell systems to elucidate the molecular and biochemical mechanisms underlying the cellular dysfunction associated with CF is crucial. The recent development of the CF pig (Rogers et al. 2008a, 2008b, 2008c) and CF ferret (Li et al. 2006; Sun et al. 2008) has added to the repertoire of animal model systems. However, long-term cell culture systems have not been developed for either of these animal models. "
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