Riechmann Veit
Medical Faculty Mannheim, Department of Cell and Molecular Biology, Heidelberg University, Ludolf-Krehl-Strasse 13-17, D-68167, Mannheim, Germany.
Methods Mol Biol. 2017;1622:185-206. doi: 10.1007/978-1-4939-7108-4_14.
In vivo RNAi in Drosophila facilitates simple and rapid analysis of gene functions in a cell- or tissue-specific manner. The versatility of the UAS-GAL4 system allows to control exactly where and when during development the function of a gene is depleted. The epithelium of the ovary is a particularly good model to study in a living animal how stem cells are maintained and how their descendants proliferate and differentiate. Here I provide basic information about the publicly available reagents for in vivo RNAi, and I describe how the oogenesis system can be applied to analyze stem cells and epithelial development at a histological level. Moreover, I give helpful hints to optimize the use of the UAS-GAL4 system for RNAi induction in the follicular epithelium. Finally, I provide detailed step-by-step protocols for ovary dissection, antibody stainings, and ovary mounting for microscopic analysis.
果蝇体内的RNA干扰技术有助于以细胞或组织特异性方式对基因功能进行简单快速的分析。UAS-GAL4系统的多功能性使得能够精确控制在发育过程中基因功能缺失的位置和时间。卵巢上皮是研究活体动物中干细胞如何维持以及其后代如何增殖和分化的一个特别好的模型。在这里，我提供了关于用于体内RNA干扰的公开可用试剂的基本信息，并描述了如何将卵子发生系统应用于在组织学水平分析干细胞和上皮发育。此外，我给出了一些有用的提示，以优化UAS-GAL4系统在卵泡上皮中诱导RNA干扰的使用。最后，我提供了卵巢解剖、抗体染色和卵巢固定以进行显微镜分析的详细分步方案。
