Generation of a CF Pig by SFHR and Nuclear Transfer

通过 SFHR 和核转移产生 CF 猪

• 批准号: 6720334
• 负责人: Dieter C Gruenert
• 金额: $ 16万
• 依托单位: CALIFORNIA PACIFIC MED CTR RES INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6720334
• 关键词: biotechnology; chloride channels; cystic fibrosis; disease /disorder model; egg /ovum; electroporation; embryogenesis; fibroblasts; flow cytometry; gene deletion mutation; gene expression; gene targeting; microinjections; model design /development; nuclear transfer; oligonucleotides; polymerase chain reaction; southern blotting; swine; tissue /cell culture

The development of transgenic mouse models for cystic fibrosis (CF) has been an important contribution to our understanding of CF and of CF transmembrane conductance regulator (CFTR) function. However, because of the dissimilarity between mouse and human anatomy and physiology, there are limitations in what can be discerned about CF pathology and in development of CF therapies. In this context, the advent of successful mammalian animal cloning by nuclear transfer has opened the door to a host of possibilities to develop large animal models for inherited diseases like CF. Recent studies investigating oligonucleotide-based genomic gene targeting suggest that some of these approaches might be effective for modifying somatic nuclei that act as the source of genetic material for nuclear transplantation. Thus it now appears possible to produce a more appropriate animal model of CF. A candidate as an alternative to the mouse model is the pig. The similarity between the human and the pig, both biochemically and physiologically, has been noted on numerous occasions and is at the heart of organ xenotransplantation. The recent sequencing of major portions of pig CFTR (pCFTR) gene has now provided the genetic information necessary to manipulate the pCFTR sequence and generate a transgenic CF animal. The small fragment homologous replacement (SFHR) gene targeting strategy is an oligonucleotide-based approach that has been previously used to modify both human and mouse CFTR by introducing the 3-bp deletion that gives rise to the AF508, CFTR mutation that predominates in the CF patient population. It is therefore well suited to generate transgenic cell lines that can be used for nuclear transplantation. This proposal will employ SFHR to modify pig fetal fibroblasts and introduce the AF508 mutation into the pCFTR. These transgenic donor cells will be clonally enriched by Fluorescence Activated Cell Sorting (FACS) and then be fused with enucleated pig oocytes that will ultimately be used for the generation of the CF pig. The transgenic donor cells will be initially screened by PCR and then evaluated by Southern blot hybridization. Transplanted oocytes will also be PCR screened and then tested in vitro for embryogenic potential. Those oocytes that have demonstrated embryogenic potential will be introduced into surrogate mothers. The development of such a transgenic CF pig will greatly enhance our ability to evaluate CF pathology and will facilitate development of new therapeutic regimens to improve the quality of life of CF patients.

囊性纤维化（CF）转基因小鼠模型的开发对我们了解CF和CF跨膜电导调节因子（CFTR）功能做出了重要贡献。然而，由于小鼠和人类解剖学和生理学的不同，在CF病理学和CF治疗的发展方面存在局限性。在这种情况下，通过核移植成功克隆哺乳动物的出现为开发大型动物模型（如CF）的可能性打开了大门。最近的研究调查了基于阿托伐他汀的 基因组基因靶向表明，这些方法中的一些可能是有效的， 作为核移植遗传物质来源的体细胞核。因此，现在似乎有可能产生更合适的CF动物模型。作为小鼠模型的替代品的候选者是猪。人类和猪在生物化学和生理学上的相似性已经在许多场合被注意到，并且是器官异种移植的核心。最近对猪CFTR（pCFTR）基因的主要部分的测序现在已经提供了操纵pCFTR序列和产生转基因CF动物所必需的遗传信息。小片段同源置换（SFHR）基因靶向策略是一种基于寡核苷酸的方法， 用于通过引入3-bp缺失来修饰人和小鼠CFTR，所述3-bp缺失产生CF患者群体中占优势的AF 508 CFTR突变。因此，它非常适合产生可用于核移植的转基因细胞系。该提案将利用SFHR修饰猪胎儿成纤维细胞，并将AF 508突变引入pCFTR。这些转基因供体细胞将通过荧光激活细胞分选（FACS）进行克隆富集，然后与去核猪卵母细胞融合，最终用于产生CF猪。首先通过PCR筛选转基因供体细胞，然后通过Southern印迹杂交进行评价。移植的卵母细胞还将进行PCR筛选，然后在体外测试胚胎发生潜力。那些已经证明具有胚胎发生潜力的卵母细胞将被引入代孕母亲。这种转基因CF猪的开发将大大提高我们评估CF病理学的能力，并将促进新的治疗方案的开发，以改善CF患者的生活质量。