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，对于功能基因组研究，大鼠是比小鼠更好的动物模型。大鼠和人类的生理和药物代谢比小鼠和人类更相似。大鼠比小鼠大，这使得许多研究更容易进行，采样也更准确。最近完成的挪威棕色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，许多可用于小鼠的基因操作技术，例如使用基因陷阱（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小鼠中都是不可能的。因为目前还没有办法有效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，效率低且不实用。这种缺陷导致我们创建人类疾病大鼠模型的能力遇到严重瓶颈。该提案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）。 Transposagen 开发了一种基于人类 L1 逆转录转座子的新技术，可产生生殖细胞中具有高随机基因突变率的动物。在 MutaRats 中，精子和卵母细胞中会发生突变，一些后代将含有单基因破坏。这些后代可用于建立真正的基因敲除大鼠系。在第一阶段，我们成功开发并表征了 MutaMice，产生了 22 只潜在的创始人大鼠。我们随后开发了类似的“变异”创始人大鼠（MutaRats），在第二阶段，我们打算表征这些老鼠，并用它们来产生具有稳定、单基因缺失（基因敲除 MutaRat 动物模型）。具体目标1是筛选现有的MutaRatFounder系以确定突变频率最高的系。然后将培育该品系以建立可用于获得特定目标 2 中的单基因敲除大鼠的繁殖群体。我们不打算表征这些品系的表型，也不打算建立个体敲除大鼠品系的繁殖群体作为这项提案的目的是建立一个包含序列插入基因破坏的精子库，即 MutaRat Germ Line Resource。在具体目标 3 中，我们将开发来自单基因敲除大鼠的冷冻精子的 MutaRat Germ Line Resource。我们估计，到 2008 年 12 月该项目结束时，我们将拥有一个包含至少 100 个不同基因敲除的精子库。该 MutaRat Germ Line Resource 将是唯一能够在大鼠中创建和快速绘制随机基因敲除图谱的技术。基因敲除大鼠将根据NIH关于生物医学研究模型生物共享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（根据需要）并分销这些产品线以及开发的其他产品。该提案的创新之处在于使用新型逆转录转座子技术来创建唯一能够在大鼠和其他哺乳动物中快速创建和定位插入缺失的系统。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具有显着优势，并产生以前在任何脊椎动物中都没有的新型基因敲除模型。 Transposagen 的 MutaRat 动物模型将为研究发病机制以及发现和开发药物化合物提供新的脊椎动物疾病模型，从而为人类健康做出贡献。