Development of New Knockout Rat Models for PKD

描述（由申请人提供）：本项目的广泛、长期目标是证明使用大鼠胚胎干细胞（ESCs）创建靶向敲除大鼠模型的可行性。大鼠ESCs的缺乏一直是对大鼠基因组进行遗传操作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效用开辟了道路。本应用程序的具体目标是通过创建Pkd1条件敲除大鼠来演示概念验证。核心假设是，大鼠胚胎干细胞将适用于已经在小鼠身上成功开发的标准敲除技术，并且靶向和特异性敲除已知导致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Pkd1大鼠敲除的基本原理是多囊肾病（PKD）是一个主要的人类健康问题，目前没有Pkd1大鼠模型存在，大多数可用的PKD啮齿动物模型并不完全模仿人类常染色体显性PKD，为旨在评估治疗方案的转化研究提供了不太理想的系统。成功培育出Pkd1基因敲除大鼠，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)在大鼠胚胎干细胞中产生大鼠Pkd1基因的靶向条件基因敲除等位基因；2)在他莫昔芬诱导控制下，建立一个携带人类Pkd1调控元件驱动的Cre重组酶转基因大鼠系；3)利用这些系建立新的PKD大鼠模型，并对它们进行批判性表征，以评估它们对人类常染色体显性PKD的模拟程度。成功完成拟议的研究将证明使用大鼠胚胎干细胞敲除大鼠感兴趣的基因以创建相关人类疾病模型的可行性。以有针对性的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的影响，广泛应用于基础研究和临床前转化研究，以评估治疗和治疗方法。

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.