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）：本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。大鼠是许多小鼠不适合的人类疾病的理想模型。大鼠是心血管和高血压疾病最相关的模型，与小鼠相比，其心率与人类更相似。与小鼠相反，大鼠和人类也共享更相似数量的细胞色素P450基因，这可能解释了为什么大鼠一直是毒理学和药理学研究的更有用的模型。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着丝粒的，这使得染色体比较与人类遗传疾病的建模更相关。然而，由于大鼠胚胎干细胞不能培养或操作，因此不能通过传统的胚胎干细胞（ES细胞）同源重组方法容易地产生大鼠突变体。替代方法效率低，成本高。虽然大鼠在过去20年里不像小鼠那样容易进行基因操作，但它确实与人类在生理、解剖和染色体上有相似之处，这使得它成为一个更相关的人类疾病模型。现在，这项提议试图通过开发用于生殖系插入诱变的超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转座子，这使得能够快速识别序列标记的突变位点。目标将集中于从三个不同的转座子家族合成超活性转座酶，每个家族都具有独特的插入特征。目前使用DNA转座子在啮齿动物中进行诱变的标准是在大鼠和小鼠中每个配子插入1-3个的速率。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，这种转座率必须大大提高。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。过度活跃的转座酶将是这项研究的直接产物，而最终的II期目标将是产生用于模拟人类疾病的大鼠突变体（MutaRats）。MutaRat的后代（MutaRat敲除大鼠模型）将不会进行表型分析或交配以产生繁殖群体，但精子将从突变雄性中分离出来，并进行低温冷冻，以便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关于共享生物医学研究模式生物的政策与学术界共享。 公共卫生关系：大约89%的用于药物使用的所有化合物在批准的最后阶段由于不可接受的副作用或缺乏疗效而失败。在进入人体药物测试的最昂贵阶段之前，显然需要在临床前测试期间在动物中更有效地筛选药物。该项目旨在采用一种新的方法，利用移动的DNA元件（或“跳跃基因”）快速、经济地生产各种各样的人类疾病小鼠和大鼠模型，这将使候选药物得到更严格的审查，并将促进更有利的人体试验。