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: 7221472
• 负责人: ERIC M OSTERTAG
• 金额: $ 68.81万
• 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 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7221472
• 关键词: Animal Model; Animals; Biological Assay; Biomedical Research; Breeding; Cells; Chemicals; Cloning; Communities; DNA; Data; Development; Disease model; Disruption; 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; Polymerase Chain Reaction; Population; Process; Production; Rat Strains; Rat Transgene; Rate; 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; desire; drug discovery; drug metabolism; embryonic stem cell; functional genomics; genetic manipulation; human disease; improved; innovation; knockout gene; male; new technology; novel; rat genome; size; 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，大鼠是比小鼠更好的功能基因组研究动物模型。大鼠和人类的生理和药物代谢比小鼠和人类更相似。大鼠比小鼠大，使得许多研究更容易进行，采样更准确。最近完成的布朗挪威大鼠基因组进一步提高了大鼠作为研究人类疾病的优秀生物体的潜力。不幸的是，许多在小鼠中可用的遗传操作技术，如用基因陷阱（基于逆转录病毒和非逆转录病毒的）随机诱变、基因敲除、基因敲入和条件突变，在大鼠中是不可能的，因为没有方法有效地收获和培养大鼠胚胎干细胞。现有的在大鼠中产生基因缺失的方法，包括克隆和化学突变，效率低下且不实用。这种缺陷导致我们在建立人类疾病大鼠模型的能力方面遇到严重瓶颈。该提案将产生的产品将是单基因敲除的大鼠，用作研究和药物发现中的人类疾病模型（MutaRat动物模型）。转座基因是一种基于人类L1反转录转座子的新技术，可以在生殖细胞中产生高随机基因突变率的动物。在MutaRat中，突变将发生在精子和卵母细胞中，一些后代将包含单基因破坏。这些后代可用于建立真正的基因敲除大鼠品系。在第一阶段，我们成功地开发和表征了MutaMice，产生了22个潜在的创始者。我们随后开发了类似的“突变体”创始者大鼠（MutaRat），在第二阶段，我们打算表征这些大鼠，并使用它们产生稳定的单基因缺失大鼠（基因敲除MutaRat动物模型）。具体目标1是筛选现有的MutaRatfounder品系以确定具有最高突变频率的品系。然后对该品系进行繁殖，以建立可用于获得特定目标2中单基因敲除大鼠的繁殖群体。我们不打算描述这些品系的表型，也不打算建立单个敲除大鼠品系的繁殖群体作为本提案的一部分，而是建立一个包含测序插入基因破坏的精子库，MutaRat生殖系资源。在具体目标3中，我们将开发单基因敲除大鼠冷冻精子的MutaRat生殖系资源。我们估计，到2008年12月这个项目结束时，我们将拥有一个至少有100个不同基因敲除的精子库。这种MutaRat生殖系资源将是唯一能够在大鼠中创建和快速绘制随机基因敲除的技术。敲除大鼠将根据NIH关于共享生物医学研究模式生物的政策提供给学术界，并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动物公司建立分销合作伙伴关系，以营销、建立和验证（必要时）并分销这些品系以及开发的其他品系。该提案的创新之处在于使用一种新型逆转录转座子技术来创建唯一能够快速创建和绘制大鼠和其他哺乳动物插入缺失的系统。与现有的小鼠诱变系统相比，该技术有望在成本和速度方面提供显着优势，并产生以前在任何脊椎动物中都不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疾病模型来研究发病机制以及发现和开发药物化合物，从而为人类健康做出贡献。