Development of New Knockout Rat Models for PKD

描述(由申请人提供)：该项目广泛的、长期的目标是证明使用大鼠胚胎干细胞(ESCs)创建有针对性的基因敲除大鼠模型的可行性。缺乏大鼠胚胎干细胞一直是通过基因操作大鼠基因组来创建特定大鼠模型的主要障碍，最近这种生物资源的可获得性为提高大鼠作为动物模型系统的实用性打开了道路。本应用程序的具体目标是通过创建PKD1条件敲除比率来演示概念验证。中心假设是，大鼠胚胎干细胞将服从已在小鼠身上成功开发的标准基因敲除技术，并且靶向和特定敲除已知导致人类疾病的基因的能力将使适当的大鼠模型得以开发。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。建立PKD1大鼠基因敲除的基本原理是，多囊肾病(PKD)是一个主要的人类健康问题，目前还不存在PKD1大鼠模型，并且大多数现有的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翻译研究提供了不太理想的系统。PKD1基因敲除大鼠的成功创造将为标准化敲除大鼠感兴趣的任何疾病基因的方法奠定基础。有三个目的被提出：1)在大鼠ES细胞中产生一个有针对性的条件基因敲除等位基因；2)在他莫昔芬的诱导控制下，建立一个携带人PKD1调控元件驱动的Cre重组酶转基因的转基因大鼠系；以及3)利用这些系建立新的大鼠PKD模型，并对其进行鉴定，以评估它们是否真实地模拟了人类常染色体显性PKD。这项拟议研究的成功完成将证明利用大鼠胚胎干细胞敲除大鼠感兴趣的基因来建立相关的人类疾病模型的可行性。有针对性地操纵大鼠基因组的能力将对大鼠作为生物医学研究的动物模型的效用产生深远的影响，在基础研究和临床前翻译研究中广泛应用于评估治疗和治疗。 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，而大鼠是特别好的模型系统，因为它们的体型(与小鼠相比)很大，而且生理上与人类相似。在这项提案中，新可用的生物试剂(大鼠胚胎干细胞)将用于创建多囊肾病(PKD)的新大鼠模型。多囊肾病是一种流行的基于遗传的疾病，估计全球约有1200万人受到影响。拟议中的实验不仅将导致研究PKD的改进动物模型，而且它们将证明通过遗传操作大鼠基因组以能够针对任何感兴趣的基因来创建新的大鼠模型来研究人类疾病的可行性。

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.