Analyzing multigenerational effects of the Piwi/piRNA pathway in zebrafish.

移动DNA元件的新整合和这些元件的扩增对真核生物基因组进化有重大贡献。然而，转座子插入在许多情况下对宿主生物是有害的。为了对抗转座子的增殖，动物种系已经发展出一种适应性的、基于RNAi的免疫系统，piwi相互作用RNA （piRNA）途径，以沉默转座子元件（te）并保持基因组完整。与其他小调控rna相比，人们对pirna的生物发生、靶沉默机制和细胞类型特异性功能知之甚少。该项目的总体目标是更多地了解pirna对TE沉默的机制以及这种沉默的跨代维持，特别是在脊椎动物中。为了分析脊椎动物piRNA的基本功能，我们将在体内建立一个独特的piRNA/靶基因系统。为此，将产生转基因斑马鱼（Danio rerio）系，表达来自EGFP修饰的te的不同pirna以及外源靶序列，生殖细胞特异性vasa::EGFP。斑马鱼的piRNA机制与黑腹果蝇和老鼠的piRNA机制非常相似。它已知的优势，如胚胎快速发育和胚胎透明，加上将新的TE拷贝引入斑马鱼基因组的成熟方法，使其成为特别适合拟议项目的模式生物。通过使用这个体内系统，我们将研究(1)独特基因组位点的piRNA合成，(2)piRNA对最近引入的转座子的反应是如何启动的，以及(3)不同Piwi途径组分在靶基因长期沉默中的可能功能。这项工作将大大增加我们对TE沉默和脊椎动物基因组完整性保护的认识。

Novel integration of mobile DNA elements and amplification of these elements contributes substantially to eukaryotic genome evolution. Nevertheless, transposon insertions can in many cases be harmful for host organisms. To counteract the proliferation of transposons, the animal germ line has developed an adaptive, RNAi based immune system, the Piwi-interacting RNA (piRNA) pathway, to silence transposable elements (TEs) and keep genomes intact. In contrast to other small regulatory RNAs, only little is known about biogenesis, target silencing mechanisms and cell type specific functions of piRNAs. The overall aim of this project is to learn more about the mechanisms of TE silencing by piRNAs and cross-generational maintenance of this silencing, especially in vertebrates.To analyze the basic functions of vertebrate piRNAs, we will set up a unique piRNA/target gene system in vivo. To this end, transgenic zebrafish (Danio rerio) lines expressing distinct piRNAs from EGFP-modified TEs as well as an exogenous target sequence, the germ cell specific vasa::EGFP, will be generated. The zebrafish piRNA machinery closely resembles the ones described for Drosophila melanogaster and mouse. Its known advantages, like fast embryonic development and transparency of embryos, combined with well-established methods to introduce new TE copies into the zebrafish genome makes it a particularly suitable model organism for the proposed project. By using this in vivo system, we are going to investigate (1) piRNA synthesis from unique genomic loci, (2) how the piRNA response to recently introduced transposons is initiated and (3) the possible functions of different Piwi pathway components in long term silencing of target genes. This work will substantially increase our knowledge about TE silencing and protection of genomic integrity in vertebrate species.