Development of a Genetic Therapy for CF in a CF Rabbit

描述（由申请人提供）：囊性纤维化（CF）转基因小鼠模型的开发对我们理解CF和CF跨膜传导调节因子（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。最近的研究调查寡核苷酸为基础的基因组基因打靶表明，这些方法中的一些可能是有效的修改体细胞核，作为核移植的遗传物质的来源。因此，现在似乎有可能产生更合适的CF动物模型。作为小鼠模型替代的候选动物是兔，其在遗传学和解剖学上比小鼠更接近于人类。最近对兔CFTR（rCFTR）基因的主要部分的测序现在已经提供了操纵rCFTR序列和产生转基因CF动物所必需的遗传信息。 小片段同源替换（SFHR）基因靶向策略是一种基于阿托伐他汀的方法，该方法先前已被用于通过引入bp缺失来修饰人和小鼠CFTR，该缺失引起了？F508，CF患者人群中占主导地位的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。这项建议将聘请SFHR引入一个？F508突变进入兔胎儿成纤维细胞的rCFTR。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，然后与去核兔卵母细胞融合，最终用于产生CF兔。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。还将对移植的卵母细胞进行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。转基因CF兔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。

DESCRIPTION (provided by applicant): 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 rabbit which is phylogenetically and anatomically more closely related to human than is the mouse. The recent sequencing of major portions of rabbit CFTR (rCFTR) gene has now provided the genetic information necessary to manipulate the rCFTR 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 bp deletion that gives rise to the ?F508, 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 introduce a ?F508 mutation into the rCFTR of rabbit fetal fibroblasts. These transgenic donor cells will be clonally enriched by Fluorescence Activated Cell Sorting (FACS) and then be fused with enucleated rabbit oocytes that will ultimately be used for the generation of a CF rabbit. 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 a transgenic CF rabbit 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.