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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    ABSTRACT: Experimental animals in biomedical research provide insights into disease mechanisms and models for determining the efficacy and safety of new therapies and for discovery of corresponding biomarkers. Although mouse and rat models are most widely used, observations in these species cannot always be faithfully extrapolated to human patients. Thus, a number of domestic species are additionally used in specific disease areas. This review summarizes the most important applications of domestic animal models and emphasizes the new possibilities genetic tailoring of disease models, specifically in pigs, provides.
    Reproduction in Domestic Animals 08/2012; 47 Suppl 4(s4):59-71. DOI:10.1111/j.1439-0531.2012.02056.x · 1.52 Impact Factor
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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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    ABSTRACT: In this unit, we describe the detailed procedure for a three-plasmid transfection method for rAAV production, and discuss its advantages, limitations, and troubleshooting techniques. We further discuss the rAAV purification process using CsCl gradients, as well as subsequent quality control methods using SDS-PAGE and real-time PCR to assess vector purity, packaging efficiency, and viral titer. Finally, we elaborate on a PCR-based strategy that can be used to discover novel AAV capsid sequences from primate tissue, which can be used to develop newer-generation rAAVs with a greater diversity of tissue tropism for clinical gene therapy.
    Current protocols in microbiology 08/2012; Chapter 14:Unit14D.1. DOI:10.1002/9780471729259.mc14d01s26
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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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    ABSTRACT: The prospect of developing large animal models for the study of inherited diseases, such as cystic fibrosis (CF), through somatic cell nuclear transfer (SCNT) has opened up new opportunities for enhancing our understanding of disease pathology and for identifying new therapies. Thus, the development of species-specific in vitro cell systems that will provide broader insight into organ- and cell-type-specific functions relevant to the pathology of the disease is crucial. Studies have been undertaken to establish transformed rabbit airway epithelial cell lines that display differentiated features characteristic of the primary airway epithelium. This study describes the successful establishment and characterization of two SV40-transformed rabbit tracheal epithelial cell lines. These cell lines, 5RTEo- and 9RTEo-, express the CF transmembrane conductance regulator (CFTR) gene, retain epithelial-specific differentiated morphology and show CFTR-based cAMP-dependent Cl(-) ion transport across the apical membrane of a confluent monolayer. Immunocytochemical analysis indicates the presence of airway cytokeratins and tight-junction proteins in the 9RTEo- cell line after multiple generations. However, the tight junctions appear to diminish in their efficacy in both cell lines after at  least 100 generations. Initial SCNT studies with the 9RTEo- cells have revealed that SV40-transformed rabbit airway epithelial donor cells can be used to generate blastocysts. These cell systems provide valuable models for studying the developmental and metabolic modulation of CFTR gene expression and rabbit airway epithelial cell biology.
    Cell and Tissue Research 02/2012; 347(2):357-67. DOI:10.1007/s00441-011-1296-1 · 3.57 Impact Factor
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