Creation of hyperactive transposons for mutagenesis in rodents

描述（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。对于许多小鼠不适合的人类疾病，大鼠是一种受欢迎的模型。大鼠是心血管和高血压疾病最相关的模型，与小鼠相比，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，这也许可以解释为什么大鼠在毒理学和药理学研究中更有用。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。与单中心小鼠基因组不同，大鼠和人类基因组主要是元中心的，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，通过传统的胚胎干细胞同源重组方法很难产生大鼠突变体。替代方法效率低下且成本高昂。虽然在过去的二十年里，大鼠还没有像小鼠那样容易地进行基因操作，但它与人类在生理、解剖和染色体上确实有相似之处，这使它成为更相关的人类疾病模型。现在，这个建议寻求通过开发用于生殖系插入突变的过度活跃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，可以快速识别序列标记的突变位点。Aims将专注于合成来自三个不同转座子家族的过度活跃转座子，每个转座子家族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在大鼠和小鼠中每个配子插入1-3次。然而，为了在商业上可行，并在每个后代中产生至少一个零等位基因，这种转位率必须大大提高。我们的目标是将突变频率提高到至少比目前用于啮齿动物突变的转座子高25倍的水平。这项研究的直接产物将是过度活跃的转座酶，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）。MutaRats的后代（MutaRat敲除大鼠模型）不会被表型化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冷冻，以备将来使用现有技术提取和受精。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。

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.