Generation of a CF Pig by SFHR and Nuclear Transfer

通过 SFHR 和核转移产生 CF 猪

• 批准号: 6878127
• 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 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6878127
• 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.

囊性纤维化转基因小鼠模型的建立为我们理解囊性纤维化及其跨膜电导调节功能做出了重要贡献。然而，由于小鼠和人类在解剖学和生理学上的不同，对CF的病理认识和治疗方法的发展都存在局限性。在这种背景下，通过核移植成功克隆哺乳动物动物的出现，为开发像CF这样的遗传性疾病的大型动物模型打开了一扇门。基于寡核苷酸的最新研究 基因组基因打靶表明，这些方法中的一些可能对修改 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一种更合适的CF动物模型。作为老鼠模型替代品的候选动物是猪。人类和猪在生化和生理上的相似性已经被多次注意到，这是异种器官移植的核心。最近对猪cftr基因主要部分的测序已经为操纵pcftr序列和产生转基因的cf动物提供了必要的遗传信息。小片段同源替换(SFHR)基因打靶策略是一种基于寡核苷酸的方法，在此之前 用于通过引入引起AF508突变的3-碱基缺失来修改人和小鼠的CFTR，该突变在CF患者群体中占主导地位。因此，它非常适合于产生可用于核移植的转基因细胞系。这项建议将使用SFHR来修饰猪胎儿成纤维细胞，并将AF508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)进行克隆浓缩，然后与去核的猪卵母细胞融合，这些卵母细胞最终将用于生成CF猪。转基因供体细胞将通过聚合酶链式反应进行初步筛选，然后进行Southern杂交鉴定。移植的卵母细胞也将进行聚合酶链式反应筛选，然后在体外测试胚胎发生潜力。那些显示出胚胎发生潜力的卵母细胞将被引入代孕母亲体内。这种转基因CF猪的研制将极大地提高我们评估CF病理的能力，并将促进新的治疗方案的开发，以改善CF患者的生活质量。

项目成果

Cftr gene targeting in mouse embryonic stem cells mediated by Small Fragment Homologous Replacement (SFHR).

小片段同源替换 (SFHR) 介导的小鼠胚胎干细胞中的 Cftr 基因靶向。

• DOI: 10.2741/2904
• 发表时间: 2008
• 期刊: Frontiers in bioscience : a journal and virtual library
• 作者: Sangiuolo,Federica;Scaldaferri,MariaLucia;Filareto,Antonio;Spitalieri,Paola;Guerra,Lorenzo;Favia,Maria;Caroppo,Rosa;Mango,Ruggiero;Bruscia,Emanuela;Gruenert,DieterC;Casavola,Valeria;DeFelici,Massimo;Novelli,Giuseppe
• 通讯作者: Novelli,Giuseppe

Gel purification of genomic DNA removes contaminating small DNA fragments interfering with polymerase chain reaction analysis of small fragment homologous replacement.

基因组 DNA 的凝胶纯化可去除干扰小片段同源替换聚合酶链反应分析的污染性小 DNA 片段。

• DOI: 10.1089/oli.2006.16.375
• 发表时间: 2006
• 期刊: Oligonucleotides
• 作者: Maurisse,Rosalie;Fichou,Yann;DeSemir,David;Cheung,Judy;Ferec,Claude;Gruenert,DieterC
• 通讯作者: Gruenert,DieterC