Novel method to create knockout rats using endonucleases and spermatagonialstem

描述（由申请方提供）：实验室大鼠是临床前药物研究中的首选啮齿动物模型，涵盖了药物开发中积累的大量知识。它们更大的尺寸有助于在小鼠中进行其他困难的手术，包括使用仪器，血液采样和手术的研究。尽管大鼠比小鼠更适合于药理学、毒理学、生理学和许多生物学测定，但缺乏适用于大鼠的基因工程技术使得小鼠成为卓越的啮齿动物模型。然而，最近出现的新的和更精确的基因靶向技术的大鼠已导致转基因大鼠的生产显着增长，这无疑将以指数速度扩大。采用转基因大鼠作为首选啮齿动物模型的限制因素是其生产所需的昂贵、低效和劳动密集型操作，导致每个转基因大鼠品系的成本高达50，000至100，000美元。因此，有必要开发更可行的大鼠基因组工程技术，从而更准确，更有效，更经济地生产转基因大鼠。Transposagen Biopharmaceuticals，Inc.是转基因大鼠模型的全球领导者，在促进这一领域的进步方面具有独特的地位。Precision BioSciences基于工程化归巢核酸内切酶开发定制基因组工程酶。这项研究提案是一项合作努力，旨在合并这两种技术，并测试使用工程化归巢核酸内切酶作为一种极其特异和有效的方法来生产转基因大鼠的可行性。在第一阶段，我们将尝试敲除大鼠Rag1基因的精原干细胞（SSC）使用Rag1特异性内切酶。然后，这些修饰的细胞将用于产生敲除Rag1突变大鼠。为了比较，我们将尝试在SSCs中使用常规基因靶向方法来实现相同的结果。在SSCs中掺入工程化核酸内切酶将大大简化敲除大鼠的生产过程，Transposagen Biopharmaceuticals和Precision BioSciences将在大规模的II期项目中合作，以产生精确工程化的高价值大鼠模型。该项目的成功将大大降低转基因大鼠的生产成本，并使学术和工业研究人员更容易获得改进的啮齿动物模型。 公共卫生相关性：实验室大鼠一直是生物医学研究的宝贵动物模型，因为它与人类生理学相似。然而，与在小鼠中产生突变相关的容易性和较低的成本导致了对基因工程小鼠模型的更大依赖，尽管这些模型中的许多不能模拟人类疾病。我们概述了一种新的策略，将我们在精原干细胞（SSCs）的专业知识与位点特异性酶技术相结合，以提高诱变效率。我们的目标是建立一个创新的转基因大鼠服务，为客户提供一个更低的成本和更有效的替代目前的转基因大鼠技术。因此，如果可行性得到证明，该项目将有利于公共卫生的许多目标，使突变的生产在大鼠模型人类疾病容易获得的研究界。

DESCRIPTION (provided by applicant): The laboratory rat is the preferred rodent model in pre-clinical drug studies and encompasses the bulk of accumulated knowledge in drug development. Their larger size facilitates procedures otherwise difficult in mice, including studies using instrumentation, blood sampling, and surgeries. Although rats are more suitable than mice for pharmacological, toxicological, physiological, and many biological assays, the absence of genetic engineering technologies suitable for use in the rat has made the mouse the preeminent rodent model. However, the recent emergence of new and more precise gene targeting techniques for the rat has resulted in significant growth in the production of genetically modified rats, which will undoubtedly expand at an exponential rate. A limiting factor in the adoption of genetically modified rats as the rodent model of choice has been the expensive, inefficient, and labor intensive manipulations required for their production, resulting in costs as high as $50,000 to $100,000 per genetically modified rat line. Consequently, it is essential to develop more viable technologies for rat genome engineering that result in more accurate, efficient, and cost-effective production of genetically modified rats. Transposagen Biopharmaceuticals, Inc. is the worldwide leader in the generation of genetically modified rat models and is uniquely positioned to facilitate advancements in this field. Precision BioSciences develops custom genome engineering enzymes based on engineered homing endonucleases. This research proposal is a collaborative effort to merge the two technologies and test the feasibility of using engineered homing endonucleases as an extremely specific and efficient method to produce genetically modified rats. In Phase I we will attempt to knockout the rat Rag1 gene in spermatagonial stem cells (SSCs) using a Rag1-specific endonuclease. These modified cells will then be used to generate knockout Rag1 mutant rats. For comparison, we will attempt to achieve the same result using conventional gene targeting methods in SSCs. The incorporation of an engineered endonuclease in SSCs should greatly streamlines the knockout rat production process, Transposagen Biopharmaceuticals and Precision BioSciences will collaborate in a large-scale Phase II project to generate precisely engineered high-value rat models. Success in this project will significantly reduce the cost of transgenic rat production and give academic and industry investigators greater access to improved rodent models. PUBLIC HEALTH RELEVANCE: The laboratory rat has been a valuable animal model for biomedical research due to its similarity to human physiology. However, the ease and lower costs associated with generating mutations in mice has lead to a greater reliance on genetically engineered mouse models despite the inability of many of these models to mimic human diseases. We outline a novel strategy that integrates our expertise in spermatogonial stem cells (SSCs) with a site-specific enzyme technology to increase efficiency of mutagenesis. We aim to establish an innovative transgenic rat service that will provide clients with a lower cost and more efficient alternative to the current genetically modified rat technology. Thus, if feasibility is demonstrated, this project would benefit many goals of public health by making the production of mutations in the rat that model human diseases readily accessible to the research community.