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

移动的DNA元件的新整合和这些元件的扩增实质上有助于真核基因组进化。然而，转座子插入在许多情况下可能对宿主生物体有害。为了抵消转座子的增殖，动物生殖系已经开发出一种适应性的、基于RNAi的免疫系统，即Piwi相互作用RNA（皮尔纳）途径，以沉默转座因子（TE）并保持基因组完整。与其他小调控RNA相比，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。本项目的总体目标是进一步了解piRNA沉默TE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皮尔纳/靶基因系统。为此，将产生表达来自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）的转基因斑马鱼（Danio rerio）系。斑马鱼的皮尔纳机制与果蝇和小鼠的piRNA机制非常相似。其已知的优势，如快速胚胎发育和胚胎的透明度，结合成熟的方法将新的TE拷贝引入斑马鱼基因组，使其成为拟议项目的特别合适的模式生物。通过使用这种体内系统，我们将研究（1）来自独特基因组位点的皮尔纳合成，（2）皮尔纳对最近引入的转座子的响应如何启动，以及（3）不同Piwi途径组分在靶基因的长期沉默中的可能功能。这项工作将大大增加我们的知识TE沉默和保护脊椎动物的基因组完整性。

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.