Department of Biology, East Carolina University, Greenville, NC, USA.
Biomedical Sciences Graduate Program, University of Virginia, Charlottesville, VA, USA.
Methods Mol Biol. 2023;2626:69-87. doi: 10.1007/978-1-0716-2970-3_4.
The ability to conduct spatially controlled RNA interference (RNAi) for gene knockdown using the UAS/Gal4 system is among the most appealing techniques available for analysis of gene function in the Drosophila ovary. While gene knockdown experiments in somatic cells in the developing organism (i.e., embryos and larvae) are effectively and commonly performed, the use of RNAi in adult ovarian cells can be hampered by the unintended deleterious effects of Gal4 expression in "off-target" developing tissues. Mosaic analysis overcomes these problems by imparting temporal and spatial control over gene manipulation, providing a useful tool to compare manipulated cells with wild-type cells in the same tissue. Here, we provide a method to utilize the UAS/Gal4 system in combination with the Flippase (FLP)-Flippase Recognition Target (FRT) system to generate positively labeled "FLP-Out" clones expressing a chosen RNAi in both the germline and the soma in the Drosophila ovary. This protocol outlines each step of the generation of clones and the selection of appropriate fly stocks and reagents, providing a guide to this powerful tool in the Drosophila genetic toolbox. These techniques allow for RNAi analysis within a specific cell type, providing an opportunity to study a variety of unique aspects of cell function that would not be possible in more traditional RNAi-based experiments.
利用 UAS/Gal4 系统进行空间控制的 RNA 干扰（RNAi）以实现基因敲低，是分析果蝇卵巢中基因功能最吸引人的技术之一。虽然在发育中的生物体（即胚胎和幼虫）的体细胞中进行基因敲低实验是有效且常用的，但 Gal4 表达在“非靶标”发育组织中可能会产生意外的有害影响，这会妨碍 RNAi 在成年卵巢细胞中的应用。通过对基因操作进行时间和空间上的控制，嵌合分析克服了这些问题，为比较同一组织中经基因操作的细胞与野生型细胞提供了有用的工具。在这里，我们提供了一种方法，可将 UAS/Gal4 系统与翻转酶（FLP）-翻转酶识别靶标（FRT）系统结合使用，以在果蝇卵巢的生殖细胞和体细胞中生成表达所选 RNAi 的阳性标记“FLP-Out”克隆。本方案概述了生成克隆的每一步以及适当的蝇株和试剂的选择，为在果蝇遗传工具包中使用这一强大工具提供了指南。这些技术允许在特定细胞类型内进行 RNAi 分析，为研究细胞功能的各种独特方面提供了机会，而这些方面在更传统的基于 RNAi 的实验中是不可能实现的。
