Genetic Dissection of Germ Cell Differentiation and Function

摘要 对生殖细胞的研究塑造了我们对许多基本过程的理解 不同的物种。生殖细胞具有许多长期以来一直令生物学家着迷的特征。这些细胞 经过减数分裂形成单倍体配子，它们非常擅长修复DNA损伤，它们 利用许多小RNA途径来沉默转座元件，它们重新编程它们的 表观基因组回到支持全能性的状态。在这里，我们建议将果蝇的卵巢用作 一个继续深入了解基因组稳定性、生殖细胞分化和细胞特异性的模型 转录调控和核糖体生物发生/周转。在过去的五年里，我们有 采用并优化了一些基于CRISPR-CAS9和重组工程的创新方法 操纵果蝇基因组。使用这些方法，我们已经变异和/或标记了100多个 果蝇性腺中丰富表达的基因。这项工作为 我们计划在未来五年所做的努力。我们将重点关注一些不同但相关的领域。我们 将继续鉴定高度保守的生殖细胞核酸肽酶(GCNA)基因和 它在保护不同物种生殖细胞完整性方面的作用。我们还将继续刻画 细胞质RBFOX1如何控制早期生殖细胞发育。我们之前的筛查工作已经 发现了少量表现为生殖细胞肿瘤形成或生殖细胞丧失的突变 表型。被破坏的基因的分子功能将使用工具和 我们手中掌握的方法。最后，我们正在开发一些创新工具，这些工具 将使我们能够更好地评估生殖细胞发育和早期核糖体的生物发生和周转 胚胎发生。我们对这项拟议的工作感到非常兴奋，并相信 这些项目将对我们理解生殖细胞生物学和其他分子产生积极的影响 影响人类健康的过程。

Summary The study of germ cells has shaped our understanding of many basic fundamental processes across different species. Germ cells share a number of features that have long fascinated biologists. These cells undergo meiosis to form haploid gametes, they are exceptionally good at repairing DNA damage, they utilize a number of small RNA pathways to silence transposable elements, and they reprogram their epigenome back to a state that supports totipotency. Here, we proposed to use the Drosophila ovary as a model to continue to gain insights into genome stability, germ cell differentiation, and the cell-specific regulation of mRNA translation and ribosome biogenesis/turnover. Over the last five years, we have adopted and optimized a number of innovative CRISPR-Cas9- and recombineering-based methods for manipulating the Drosophila genome. Using these approaches, we have mutated and/or tagged over 100 genes that exhibit enriched expression in Drosophila gonads. This work provides a solid foundation for our planned efforts over the next five years. We will focus on a number of different but related areas. We will continue to characterize the highly conserved Germ Cell Nuclear Acidic Peptidase (GCNA) gene and its function in protecting the integrity of germ cells across species. We will also continue to characterize how cytoplasmic Rbfox1 controls early germ cell development. Our previous screening efforts have identified a small number of mutations that exhibit germ cell tumor formation or germ cell loss phenotypes. The molecule function of the disrupted genes will be characterized using the tools and methods we have in hand. Lastly, we are in the process of generating a number of innovative tools that will allow us to better assess ribosome biogenesis and turnover during germ cell development and early embryogenesis. We are very excited by this proposed work and believe the successful completion of these projects will have a positive impact on our understanding of germ cell biology and other molecular processes that impact human health.