Novel method to create knockout rats using endonucleases and spermatagonialstem

使用核酸内切酶和精原干细胞创建基因敲除大鼠的新方法

基本信息

批准号：
8201328
负责人：
ERIC M OSTERTAG
金额：
$ 34.2万
依托单位：
TRANSPOSAGEN BIOPHARMACEUTICALS, INC.
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-05 至 2013-09-04
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8201328
关键词：
Adoption Agreement Animal Model Biological Assay Biological Products Biomedical Research Blood specimen Cell Line Cell Separation Cells Client Clone Cells Communities Custom DNA DNA Double Strand Break Deletion Mutation Diphtheria Toxin Embryo Engineering Enzymes Event Feasibility Studies Female Frameshift Mutation Frequencies Gene Targeting Generations Genes Genetic Genetic Engineering Genetic Recombination Genetically Engineered Mouse Genome engineering Genomics Goals Growth Homing Hour Human In Vitro Industry Knock-out Knowledge Laboratory Animals Laboratory Rat Lead Libraries Licensing Measures Mediating Methods Microinjections Modeling Mus Mutagenesis Mutation Neomycin Operative Surgical Procedures Pharmaceutical Preparations Phase Physiological Physiology Plasmids Polymerase Positioning Attribute Procedures Process Production Public Health RAG1 gene Rag1 Mouse Rat Cell Line Rattus Reliance Research Research Personnel Research Proposals Resistance Rodent Model Services Site Stem cells Sterility Study models Techniques Technology Testing Testis Transfection Transgenic Organisms Transplantation Validation antibiotic G 418 arm base cost cost effective drug development endonuclease hepatoma cell homologous recombination human disease improved innovation instrumentation male mouse model mutant new technology novel novel strategies pre-clinical pup rat genome success transmission process vector

项目摘要

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.

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，包含药物开发中积累的大量知识。它们较大的尺寸有利于在小鼠中进行原本困难的手术，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药理学、毒理学、生理学和许多生物测定，但由于缺乏适合在大鼠中使用的基因工程技术，使得小鼠成为卓越的啮齿动物模型。然而，最近出现的新的、更精确的大鼠基因靶向技术导致转基因大鼠的产量显着增长，毫无疑问将以指数速度增长。采用转基因大鼠作为啮齿动物模型的一个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、更高效、更经济地生产转基因大鼠。 Transposagen Biopharmaceuticals, Inc. 是转基因大鼠模型生成领域的全球领导者，在促进该领域的进步方面具有独特的优势。 Precision BioSciences 开发基于工程化归巢核酸内切酶的定制基因组工程酶。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生产转基因大鼠的极其具体和有效的方法的可行性。在第一阶段，我们将尝试使用 Rag1 特异性核酸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被用来产生敲除 Rag1 突变体的大鼠。为了进行比较，我们将尝试在 SSC 中使用传统的基因打靶方法获得相同的结果。在 SSC 中纳入工程核酸内切酶将大大简化基因敲除大鼠的生产过程，Transposagen Biopharmaceuticals 和 Precision BioSciences 将在大规模 II 期项目中合作，以生成精确工程设计的高价值大鼠模型。该项目的成功将显着降低转基因大鼠的生产成本，并使学术和工业研究人员有更多机会获得改进的啮齿动物模型。公共健康相关性：实验室大鼠由于其与人类生理学的相似性而成为生物医学研究的有价值的动物模型。然而，尽管许多模型无法模拟人类疾病，但与在小鼠中产生突变相关的简便性和较低的成本导致了对基因工程小鼠模型的更大依赖。我们概述了一种新策略，将我们在精原干细胞（SSC）方面的专业知识与位点特异性酶技术相结合，以提高诱变效率。我们的目标是建立创新的转基因老鼠服务，为客户提供比当前转基因老鼠技术更低成本、更有效的替代方案。因此，如果可行性得到证实，该项目将通过使研究界能够轻松地在大鼠中产生模拟人类疾病的突变，从而有利于公共卫生的许多目标。