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（由申请人提供）：该项目旨在通过基于转座子的诱变方法快速产生大鼠突变体。大鼠是治疗多种人类疾病的首选模型，而小鼠不适合这些疾病。大鼠是心血管和高血压疾病最相关的模型，与小鼠相比，其心率更接近人类。与小鼠不同，大鼠和人类的细胞色素 P450 基因数量也更为相似，这或许可以解释为什么大鼠是毒理学和药理学研究中更有用的模型。大鼠也是糖尿病、关节炎、行为障碍（包括毒瘾）和脑成像的首选模型。与近端着丝粒小鼠基因组不同，大鼠和人类基因组主要是中间着丝粒，使得染色体比较与人类遗传疾病建模更相关。然而，通过传统的胚胎干（ES）细胞同源重组方法无法轻易产生大鼠突变体，因为大鼠ES细胞无法培养或操作。替代方法效率低下且成本高昂。虽然大鼠在过去二十年中不像小鼠那样容易进行基因操作，但它确实与人类具有生理、解剖和染色体相似性，这使得它成为更相关的人类疾病模型。现在，该提案寻求通过开发用于种系插入诱变的高活性转座子来快速产生大鼠突变体。这种方法使用随机整合的转座子，能够快速识别序列标记的突变位点。该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，每个转座酶都具有独特的插入特性。目前使用 DNA 转座子对啮齿动物进行诱变的标准是大鼠和小鼠中每个配子 1-3 次插入。然而，为了在商业上可行并在每个后代中产生至少一个无效等位基因，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诱变的转座子至少高 25 倍的水平。高活性转座酶将是这项研究的直接产品，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（MutaRats）。 MutaRats（MutaRat 敲除大鼠模型）的后代不会进行表型分析或交配以产生繁殖群体，但将从突变雄性中分离精子并低温冷冻，以供将来使用现有技术进行检索和受精。突变动物将由国家大鼠资源和研究中心分发，并根据 NIH 生物医学研究模型生物共享政策与学术界共享。 公共健康相关性：在所有药物用途测试化合物中，约有 89% 由于不可接受的副作用或缺乏疗效而在批准的最后阶段失败。在进入人类药物测试最昂贵的阶段之前，显然需要在临床前测试期间在动物身上更有效地筛选药物。该项目旨在采用一种使用移动DNA元件（或“跳跃基因”）的新方法，快速、经济地生产多种人类疾病的小鼠和大鼠模型，这将能够对候选药物进行更严格的审查，并促进在人​​类中进行更有利的测试。