Novel method to create knockout rats using endonucleases and spermatagonialstem

描述（由申请人提供）：实验室大鼠是临床前药物研究中首选的啮齿动物模型，包含了药物开发中积累的大部分知识。它们更大的尺寸有助于在小鼠身上进行其他困难的程序，包括使用仪器、血液采样和手术的研究。虽然大鼠比小鼠更适合进行药理学、毒理学、生理学和许多生物试验，但由于缺乏适合在大鼠身上使用的基因工程技术，使小鼠成为卓越的啮齿动物模型。然而，最近出现了新的和更精确的大鼠基因靶向技术，导致转基因大鼠的生产显著增长，无疑将以指数速度增长。采用转基因老鼠作为啮齿类动物模型的一个限制因素是，它们的生产需要昂贵、低效和劳动密集型的操作，导致每条转基因老鼠的成本高达5万到10万美元。因此，必须开发更可行的大鼠基因组工程技术，从而更准确、更高效、更经济地生产转基因大鼠。转座生物制药公司是一代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。精密生物科学开发定制基因组工程酶基于工程归巢内切酶。这项研究计划是一项合作努力，将这两种技术结合起来，并测试使用工程归巢内切酶作为一种极其特定和有效的方法来生产转基因大鼠的可行性。在第一阶段，我们将尝试使用Rag1特异性内切酶敲除大鼠精原干细胞（ssc）中的Rag1基因。这些经过修饰的细胞将用于产生敲除的Rag1突变大鼠。为了比较，我们将尝试在ssc中使用传统的基因靶向方法获得相同的结果。在ssc中加入工程核酸内切酶将大大简化敲除大鼠的生产过程，转座生物制药公司和精密生物科学公司将在一个大规模的二期项目中合作，以产生精确工程的高价值大鼠模型。这个项目的成功将大大降低转基因大鼠生产的成本，并使学术界和工业界的研究人员更容易获得改良的啮齿动物模型。

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.