Creation of hyperactive transposons for mutagenesis in rodents

创建用于啮齿动物诱变的高活性转座子

• 批准号: 7670115
• 负责人: ERIC M OSTERTAG
• 金额: $ 29.56万
• 依托单位: TRANSPOSAGEN BIOPHARMACEUTICALS, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7670115
• 关键词: Adverse effects; Alleles; Amino Acid Substitution; Animal Model; Animals; Arthritis; Attenuated; Behavior Disorders; Biological Assay; Biomedical Research; Brain imaging; Breeding; Cardiovascular Models; Cell Line; Cells; Characteristics; Chemicals; Cloning; Code; Communities; Complement; Consensus; Culicidae; Custom; Cytochrome P450; DNA; DNA Shuffling; DNA Transposons; Development; Diabetes Mellitus; Disease; Drug Addiction; Drug usage; Elements; Engineering; Event; Family; Family suidae; Fertilization; Flow Cytometry; Freezing; Frequencies; Future; Gene Deletion; Gene Delivery; Gene Transfer Techniques; Generations; Genes; Genetic Recombination; Genome; Genomics; Germ Cells; Germ Lines; Goals; Heart Rate; Hela Cells; Hereditary Disease; Human; Human Genetics; Human Genome; Human Pathology; Hyperactive behavior; Hypertension; Individual; Insertional Mutagenesis; Jumping Genes; Knock-out; Libraries; Malaria; Mammalian Cell; Methods; Mission; Modeling; Modification; Mus; Mutagenesis; Mutation; Organism; Partner in relationship; Paste substance; Performance; Pharmacologic Substance; Pharmacology and Toxicology; Phase; Phenotype; Phylogenetic Analysis; Physiological; Physiology; Policies; Preclinical Drug Evaluation; Preclinical Testing; Production; Property; Proteins; RNA; Rattus; Recombinants; Research; Resistance; Resources; Retrieval; Retrotransposon; Rodent; Ruminants; Sequence Alignment; Series; Site; Sleeping Beauty; Small Business Innovation Research Grant; Sperm Banks; Staging; System; Techniques; Technology; Testing; Time; Toxicology; Transgenic Animals; Transgenic Organisms; Transposase; Tribolium; United States National Institutes of Health; Variant; Work; Yeasts; base; cost; design; directed evolution; drug candidate; drug development; drug testing; embryonic stem cell; falls; genetic manipulation; genome-wide; high throughput screening; homologous recombination; human disease; improved; in vitro Assay; knockout gene; male; meetings; mouse genome; mouse model; mutant; novel; offspring; polypeptide; public health relevance; rat genome; sperm cell; therapeutic gene; tool; transposon/insertion element; vector

DESCRIPTION (provided by applicant): This project aims to rapidly produce rat mutants through a transposon-based method of mutagenesis. The rat is a favored model for many types of human disease for which mice are not suitable. The rat is the most relevant model for cardiovascular and hypertension disease, with a heart rate much more similar to that of humans compared to mice. As opposed to the mouse, rats and humans also share a more similar number of cytochrome P450 genes, perhaps explaining why the rat has been a more useful model for toxicology and pharmacology studies. The rat is also a favored model for diabetes, arthritis, behavioral disorders (including drug addiction), and brain imaging. Unlike the acrocentric mouse genome, the rat and human genomes are predominantly metacentric, making chromosomal comparisons more relevant for modeling human genetic diseases. However, rat mutants cannot be easily generated through traditional methods of homologous recombination in embryonic stem (ES) cells, because rat ES cells cannot be cultured or manipulated. Alternative methods are inefficient and costly. While the rat has not had the ease of genetic manipulation that the mouse has had over the past twenty years, it certainly possesses physiological, anatomical, and chromosomal similarities to humans that make for a more relevant model of human disease. Now, this proposal seeks to rapidly produce rat mutants through the development of hyperactive transposons for germ line insertional mutagenesis. This approach uses randomly integrating transposons, which enables the rapid identification of sequence-tagged mutation sites. The Aims will focus on synthesizing hyperactive transposases from three different families of transposons, each with unique insertion characteristics. The current standard for mutagenesis in rodents using DNA transposons is a rate of 1-3 insertions per gamete in rats and mice. However, to be commercially viable and produce at least one null allele in each offspring, this transposition rate must be substantially improved. Our goal is to increase the mutation frequency to a level at least 25X greater than the transposons currently used for rodent mutagenesis. Hyperactive transposases will be the direct product from this study, while the ultimate Phase II goal will be the generation of rat mutants (MutaRats) for modeling human disease. Offspring of MutaRats (MutaRat Knockout Rat Models) will not be phenotyped or mated to produce a breeding colony, but sperm will be isolated from mutant males and cryogenically frozen for future retrieval and fertilization using already existing technology. Mutant animals will be distributed by the National Rat Resource and Research Center and shared with the academic community according to NIH policies for sharing model organisms for biomedical research. PUBLIC HEALTH RELEVANCE: About 89% of all compounds tested for drug use fail during the final stages of approval due to unacceptable side effects or a lack of efficacy. There is a clear need to screen drugs more effectively in animals during preclinical testing before entering the most expensive phases of drug testing in humans. This project is designed to employ a new method using mobile DNA elements (or "jumping genes") for the rapid and economical production of a large variety of mouse and rat models of human disease, which will enable a greater scrutiny of candidate drugs and will facilitate more favorable testing in humans.

描述(申请人提供)：该项目旨在通过基于转座子的诱变方法快速产生老鼠突变体。对于许多类型的人类疾病，老鼠是一个受欢迎的模型，而老鼠不适合。大鼠是心血管和高血压疾病最相关的模型，与小鼠相比，大鼠的心率更接近人类。与小鼠相反，大鼠和人类的细胞色素P450基因数量也更相似，这可能解释了为什么大鼠一直是毒理学和药理学研究更有用的模型。大鼠也是糖尿病、关节炎、行为障碍(包括药物成瘾)和脑成像的理想模型。与小鼠的顶端着丝粒基因组不同，大鼠和人类的基因组主要是中着丝粒的，这使得染色体比较在模拟人类遗传病方面更具相关性。然而，由于大鼠胚胎干细胞不能培养或操纵，通过传统的同源重组方法很难在胚胎干细胞中产生大鼠突变。其他方法效率低，成本高。虽然在过去的二十年里，老鼠没有老鼠那么容易进行基因操作，但它肯定拥有与人类在生理、解剖和染色体上的相似之处，这有助于建立一个更相关的人类疾病模型。现在，这项提议寻求通过开发用于生殖系插入突变的超活性转座子来快速产生大鼠突变。这种方法使用随机整合转座子，从而能够快速识别序列标记的突变位点。这些目标将集中于从三个不同的转座子家族合成高活性转座酶，每个转座子家族都有独特的插入特性。目前使用DNA转座子对啮齿动物进行突变的标准是在大鼠和小鼠的每个配子中插入1-3个插入子。然而，为了在商业上可行，并在每个后代中产生至少一个空等位基因，必须大幅提高这种转座率。我们的目标是将突变频率提高到比目前用于啮齿动物突变的转座子高至少25倍的水平。过度活跃的转座酶将是这项研究的直接产物，而第二阶段的最终目标将是产生用于模拟人类疾病的大鼠突变体(Mutarat)。突变大鼠(突变大鼠模型)的后代将不会进行表型鉴定或交配以产生繁殖群体，但将从突变的雄性精子中分离出精子，并将其冷冻起来，以供未来使用现有技术进行取回和受精。突变动物将由国家老鼠资源和研究中心分发，并根据NIH共享生物医学研究模式生物的政策与学术界共享。 与公共卫生相关：在所有药物使用测试中，大约89%的化合物由于不可接受的副作用或缺乏疗效而在批准的最后阶段不合格。在进入人类最昂贵的药物测试阶段之前，显然需要在临床前试验期间在动物身上更有效地筛选药物。该项目旨在使用一种新的方法，使用可移动的DNA元件(或“跳跃基因”)来快速和经济地生产各种人类疾病的小鼠和大鼠模型，这将使对候选药物进行更严格的审查，并将促进更有利的人体测试。