L1 retrotransposon-based mutagenesis for rat models of human diseases

基于 L1 逆转录转座子的人类疾病大鼠模型诱变

• 批准号: 7755324
• 负责人: ERIC M OSTERTAG
• 金额: $ 18.99万
• 依托单位: TRANSPOSAGEN BIOPHARMACEUTICALS, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-14 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7755324
• 关键词: Animal Model; Animals; Biological Assay; Biomedical Research; Breeding; Cells; Chemicals; Cloning; Communities; DNA; Data; Development; Disease model; Exhibits; Exons; Freezing; Frequencies; Gene Deletion; Gene Mutation; Generations; Genes; Genetic; Germ; Germ Cells; Germ Lines; Harvest; Health; Human; Individual; Inherited; Insertional Mutagenesis; Introns; Knock-out; Laboratory Animals; Location; Mammals; Maps; Marketing; Methods; Modeling; Mus; Mutagenesis; Mutation; Oocytes; Organism; Pathogenesis; Pathology; Pattern; Pharmacologic Substance; Phase; Phenotype; Physiology; Policies; Population; Process; Production; Rat Strains; Rat Transgene; Rattus; Rattus norvegicus; Research; Resources; Retrotransposition; Retrotransposon; Sampling; Speed; Sperm Banks; System; Tail; Techniques; Technology; Testing; Transgenes; United States National Institutes of Health; base; cost; drug discovery; drug metabolism; embryonic stem cell; functional genomics; genetic manipulation; human disease; improved; innovation; knockout gene; male; new technology; novel; offspring; rat genome; sperm cell; success

DESCRIPTION (provided by applicant): In many aspects the rat is a better animal model than the mouse for functional genomic studies. The physiology and drug metabolism of rats and humans are more similar than that of mice and humans. The rat is larger than the mouse, making many studies easier to perform and sampling more accurate. The recent completion of the Brown Norway rat genome improves further the potential of the rat as an excellent organism for studying human diseases. Unfortunately, many genetic manipulation techniques available in the mouse such as random mutagenesis with a gene trap (both retroviral-based and non-retroviral-based), gene knock- outs, gene knock-ins, and conditional mutations have not been possible in the rat because there is no way to effectively harvest and culture rat embryonic stem cells. Existing methods for producing gene deletions in rats, including cloning and chemical mutation, are inefficient and not practical. This deficiency leads to a severe bottleneck in our ability to create rat models of human disease. The product that will arise from this proposal will be rats with single gene knockouts for use as models of human disease in research and drug discovery (MutaRat Animal Models). Transposagen has developed a novel technology, based on human L1 retrotransposons, to generate animals with a high rate of random gene mutations in germ cells.In MutaRats, mutagenesis will occur in sperm and oocytes and some offspring will contain single gene disruptions. These offspring can be used to establish authentic gene knockout rat lines. In Phase I we successfully developed and characterized MutaMice, generating 22 potential founders.We have subsequently developed similar "mutator" founder rats (MutaRats), and in Phase II, we intend to characterize these rats, and use them to generate rats with stable, singe gene deletions (gene knockout MutaRat Animal Models). Specific aim 1 isto screen existing MutaRatfounder lines to determine the line with the highest frequency of mutation. This line will then be bred to establish a breeding colony that can be used to obtain single gene knockout rats in Specific Aim 2. We do not intend to characterize the phenotypes of these lines, nor to establish breeding colonies of individual knockout rat linesas part of this proposal, but instead to establish a sperm bank comprising sequenced insertional gene disruptions, the MutaRat Germ Line Resource. In Specific Aim 3 we will develop the MutaRat Germ Line Resourceof cryopreserved sperm from rats with single gene knockouts. We estimate that we will have a sperm bank with at least 100 different gene knockouts by the endof this project in December, 2008. This MutaRat Germ Line Resource will be the only technology capable of creating and rapidly mapping random gene knockouts in rats. Knockout ratswill be provided to the academic community in accordance with the NIH policy on sharing of model organisms for biomedical research and will be distributed by the National Rat Resource and Research Center. In Phase III, we will establish a distribution partnership with an existing laboratory animal company to market, establish and validate (as necessary), and distribute these lines, and others as they are developed. The innovation of this proposal is the use of a novel retrotransposon technology to create the only system able to rapidly create and map insertional deletions in the rat and other mammals. This technology is expected to offer significant advantages in cost and speed when compared with existing mutagenesis systems available in mice, and to generate novel gene knockout models that have not previously been available in any vertebrate. Transposagen's MutaRat Animal Models will contribute to human health by providing new vertebrate models of disease for studying pathogenesis and for discovery and development of pharmaceutical compounds.

描述（由申请人提供）：在许多方面，大鼠比小鼠在功能基因组研究中都是更好的动物模型。大鼠和人类的生理和药物代谢比小鼠和人类更相似。大鼠大于小鼠，使许多研究更易于执行和采样。棕色挪威大鼠基因组的最近完成，进一步提高了大鼠作为研究人类疾病的出色生物的潜力。不幸的是，在小鼠中可用的许多遗传操纵技术，例如具有基因陷阱的随机诱变（基于逆转录病毒和非逆转录病毒的随机诱变），基因敲除，基因敲击和有条件的突变在大鼠中无法有效，因为没有办法有效地收获和培养的大鼠胚胎胚胎干细胞。现有的在包括克隆和化学突变在内的大鼠中产生基因缺失的方法是效率低下且不实用的。这种缺乏导致我们创建大鼠人类疾病模型的能力的严重瓶颈。该提案将产生的产品将是带有单基因敲除的大鼠，可作为研究和药物发现中的人类疾病模型（Mutarat动物模型）。 Transposagen开发了一种基于人L1逆转座子的新技术，以产生具有较高随机基因突变的动物。这些后代可用于建立真实的基因敲除大鼠线。在第一阶段，我们成功地开发并表征了Mutamice，产生了22个潜在的创始人。我们随后开发了类似的“突变器”创始人大鼠（Mutarats），在第二阶段，我们打算表征这些大鼠，并使用它们来产生具有稳定的，Singe Gene deletions（Gene敲除Mutarat动物模型）的稳定的大鼠。特定的目标1 iSTO屏幕现有的mutaratfounder线，以确定最高突变频率的线。然后将构成这条线以建立一个可以在特定目标中获得单个基因敲除大鼠的繁殖菌落。我们无意表征这些线条的表型，也不是建立该提案中单个敲除大鼠线的繁殖菌落，而是要建立一个由序列的插入基因的插入基因拆卸，而是建立一个精子银行。在特定的目标3中，我们将开发来自带有单基因敲除大鼠的冷冻保存精子的mutarat种系资源。我们估计，该项目将在2008年12月拥有该项目至少有100个不同基因敲除的精子库。该Mutarat种系资源将是唯一能够在大鼠中创建和快速映射随机基因敲除的技术。根据NIH共享生物医学研究模型生物的NIH政策，将为学术界提供淘汰赛，并将由国家大鼠资源和研究中心分发。在第三阶段，我们将与现有的实验室动物公司建立分销合作伙伴关系，以营销，建立和验证（必要），并在开发过程中分发这些线路。该提案的创新是使用一种新型的逆转录座位技术来创建唯一能够在大鼠和其他哺乳动物中迅速创建和映射插入删除的系统。与小鼠现有的诱变系统相比，该技术有望在成本和速度上具有显着优势，并生成以前在任何脊椎动物中尚未可用的新型基因敲除模型。 Transposagen的Mutarat动物模型将通过提供新的脊椎动物模型来研究发病机理，发现和开发药物化合物，从而有助于人类健康。