Abstract
Creating a CFTR Knockout Ferret Model by Nuclear Transfer Cloning
Biology of reproduction, Vol.78(Suppl_1), pp.55-55
05/01/2008
DOI: 10.1093/biolreprod/78.s1.55b
Abstract
Somatic cell gene targeting combined with nuclear transfer cloning holds tremendous potential for the creation of larger and potentially superior, animal models of human diseases. Mice deficient for the cystic fibrosis transmembrane conductance regulator (CFTR) protein fail to reproduce key pathologies associated with cystic fibrosis (CF) in human patients. The domestic ferret (Mustela putorius furo) is an attractive alternative species for modeling CF, given its high level of conservation in lung biology with humans. We previously cloned ferrets from cumulus cells using somatic cell nuclear transfer (SCNT) technology. However, fibroblasts are the cell of choice for gene targeting and SCNT methods have not been developed in the ferret using this donor cell type. Therefore, our objective is to develop fibroblast-based SCNT in the ferret and to produce a CFTR gene deficient CF model animal. Our first step was to clone ferrets from non-targeted primary fetal fibroblasts. This required optimization of the age of recipient oocytes, media alterations to reduce embryo stress during manipulation, and determination of the timing of oocyte activation to more efficiently promote nuclear remodeling. SCNT and subsequent embryo transfer (ET) resulted in a 66.7% rate of pregnancy initiation and normal fetal development in 2.2% of reconstructed embryos. Primary ferret fibroblasts derived from a male E28 fetus were then infected with a recombinant adeno-associated virus (rAAV-2) to target the CFTR gene. Three CFTR-postitive knock-out cell clones were identified by PCR. However, CFTR-targeted ferret fibroblast clones senesce rapidly during expansion following the initial round of PCR screening. To rejuvenate and expand these candidate CFTR-targeted clones for Southern blot confirmation of the targeting events, we performed several rounds of SCNT on three independent PCR-positive clones. Six 21-day ferret fetuses were produced. One of the three original senescent fibroblast lines that was used for SCNT gave rise to secondary fetal fibroblasts with a "clean" CFTR gene-targeting event as shown by Southern blotting. This SCNT-rejuvenated, CFTR-targeted, fibroblast clone was then used in a final cloning step. Four hundred and seventy-two reconstructed embryos were transferred into 11 recipient Jills from which we obtained 8 healthy male ferret clones heterozygous for an exon 10 disruption in the CFTR gene. These data demonstrate that rAAV-mediated gene targeting, coupled with SCNT cloning, may be of significant utility in developing new animal models for CF and other human genetic diseases.
Details
- Title: Subtitle
- Creating a CFTR Knockout Ferret Model by Nuclear Transfer Cloning
- Creators
- Xingshen SunZiying YanYaling YiZiyi LiDiana LeiChristopher S. RogersJuan ChenMichael J. WelshGregory H. LenoJohn F Engelhardt
- Resource Type
- Abstract
- Publication Details
- Biology of reproduction, Vol.78(Suppl_1), pp.55-55
- DOI
- 10.1093/biolreprod/78.s1.55b
- ISSN
- 0006-3363
- eISSN
- 1529-7268
- Language
- English
- Date published
- 05/01/2008
- Academic Unit
- Pulmonary, Critical Care, and Occupational Medicine; Radiation Oncology; Molecular Physiology and Biophysics; Neurosurgery; Fraternal Order of Eagles Diabetes Research Center; Roy J. Carver Department of Biomedical Engineering; Internal Medicine; Anatomy and Cell Biology; Neurology
- Record Identifier
- 9984259441802771
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