Development of New Knockout Rat Models for PKD

描述（由申请人提供）：该项目的广泛，长期目标是证明使用大鼠胚胎干细胞（ESC）创建靶向敲除大鼠模型的可行性。大鼠胚胎干细胞的缺乏一直是遗传操纵大鼠基因组以创建特定大鼠模型的主要障碍，最近这种生物资源的可用性为增加大鼠作为动物模型系统的实用性开辟了道路。本申请的具体目的是通过创建Pkd1条件性敲除大鼠来证明概念验证。核心假设是大鼠ES细胞将服从已在小鼠中成功开发的标准敲除技术，并且靶向和特异性敲除已知引起人类疾病的基因的能力将允许开发适当的大鼠模型。此外，这些大鼠模型将为研究疾病和测试治疗方法提供改进的工具。创建Pkd 1敲除大鼠的基本原理是多囊肾病（PKD）是一个主要的人类健康问题，目前不存在Pkd 1大鼠模型，并且大多数可用的PKD啮齿动物模型不完全模拟人类常染色体显性PKD，为旨在评价治疗方案的转化研究提供了不太理想的系统。Pkd1基因敲除大鼠的成功建立将为敲除大鼠中任何感兴趣的疾病基因的方法标准化奠定基础。提出了三个目标：1）在大鼠ES细胞中产生大鼠Pkd 1基因的靶向条件基因敲除等位基因，2）产生携带Cre重组酶转基因的转基因大鼠品系，所述Cre重组酶转基因由在他莫昔芬的诱导控制下的人PKD 1调节元件驱动，和3）使用这些品系产生PKD的新大鼠模型，并对它们进行严格表征以评估它们模拟人常染色体显性PKD的忠实程度。成功完成拟议的研究将证明使用大鼠ES细胞敲除大鼠中感兴趣的基因以创建人类疾病的相关模型的可行性。以靶向方式操纵大鼠基因组的能力将对大鼠作为生物医学研究动物模型的效用产生深远影响，广泛应用于基础研究和临床前转化研究以评估治疗和疗法。 公共卫生相关性：模拟人类疾病的动物模型是研究疾病过程和评估治疗方法的重要工具，大鼠是特别好的模型系统，因为它们的尺寸大（与小鼠相比）和与人类的生理相似性。在这项提案中，新获得的生物试剂（大鼠胚胎干细胞）将用于创建一种新的多囊肾病（PKD）大鼠模型，这是一种普遍的遗传性疾病，估计影响全球约1200万人。拟议的实验不仅将导致改进的动物模型来研究PKD，而且它们将证明遗传操纵大鼠基因组的可行性，以便能够靶向任何感兴趣的基因，从而创建新的大鼠模型来研究人类疾病。

DESCRIPTION (provided by applicant): The broad, long-term goal of this project is to demonstrate the feasibility of using rat embryonic stem cells (ESCs) to create targeted knock-out rat models. The lack of rat ESCs has been a major impediment to genetically manipulating the rat genome to create specific rat models and the recent availability of this biological resource opens the way to increasing the rat's utility as an animal model system. The specific objective of this application is to demonstrate proof-of-concept by creating a Pkd1 conditional knock-out rat. The central hypothesis is that rat ES cells will be amenable to standard knock-out technology that has been developed successfully in the mouse and that the ability to target and specifically knock-out genes known to cause human disease will allow appropriate rat models to be developed. In addition, these rat models will provide improved tools for studying disease and for testing therapeutics. The rationale for creating a Pkd1 rat knock-out is that polycystic kidney disease (PKD) is a major human health issue, no Pkd1 rat models currently exist and most available rodent models of PKD do not entirely mimic human autosomal dominant PKD providing less than optimal systems for translational studies aimed at evaluating treatment options. Successful creation of a Pkd1 knock-out rat will set the stage for standardizing the methodology for knocking out any disease gene of interest in the rat. Three Aims are proposed: 1) produce a targeted conditional gene knock-out allele of the rat Pkd1 gene in rat ES cells, 2) create a transgenic rat line carrying a transgene for Cre recombinase driven by human PKD1 regulatory elements under inducible control of tamoxifen, and 3) use these lines to create new rat models for PKD and critically characterize them to evaluate how faithfully they mimic human autosomal dominant PKD. Successful completion of the proposed studies will demonstrate the feasibility of using rat ES cells to knock-out genes of interest in the rat to create relevant models of human disease. The ability to manipulate the rat genome in a targeted manner will have a profound impact on the utility of rats as animal models for biomedical research, with widespread applications for both basic research studies and pre-clinical translational studies to evaluate treatments and therapeutics. PUBLIC HEALTH RELEVANCE: Animal models that mimic human disease are important tools for studying disease processes and evaluating therapeutic treatments and rats are particularly good model systems due to their large size (compared to mice) and physiological similarities to humans. In this proposal, newly available biological reagents (rat embryonic stem cells) will be used to create a new rat model for polycystic kidney disease (PKD), a prevalent genetically- based disorder estimated to affect an estimated 12 million individuals worldwide. The proposed experiments will not only result in improved animal models to study PKD but they will demonstrate the feasibility of genetically manipulating the rat genome to be able to target any gene of interest in order to create new rat models to study human disease.