Department of Cell and Developmental Biology, University of Michigan.
Cellular and Molecular Biology Program, University of Michigan.
J Vis Exp. 2024 Jan 26(203). doi: 10.3791/64729.
The study of protein subcellular localization, dynamics, and regulation in live cells has been profoundly transformed by the advent of techniques that allow the tagging of endogenous genes to produce fluorescent fusion proteins. These methods enable researchers to visualize protein behavior in real time, providing valuable insights into their functions and interactions within the cellular environment. Many current gene tagging studies employ a two-step process where visible markers, such as eye color changes, are used to identify genetically modified organisms in the first step, and the visible marker is excised in the second step. Here, we present a one-step protocol to perform precise and rapid endogenous gene tagging in Drosophila melanogaster, which enables screening for engineered lines without the visible eye marker, offering a significant advantage over past methods. To screen for successful gene-tagging events, we employ a PCR-based technique to genotype individual flies by analyzing a small segment from their middle leg. Flies that pass the screening criteria are then used to produce stable stocks. Here, we detail the design and construction of CRISPR editing plasmids and methods for screening and confirmation of engineered lines. Together, this protocol improves the efficiency of endogenous gene tagging in Drosophila significantly and enables studies of cellular processes in vivo.
该研究的蛋白质亚细胞定位，动态和调节在活细胞中已经深刻转变的到来的技术，允许标记的内源性基因产生荧光融合蛋白。这些方法使研究人员能够实时可视化蛋白质的行为，提供有价值的见解，其功能和相互作用在细胞环境中。许多当前的基因标记研究采用两步法，其中可见的标记，如眼睛颜色的变化，用于在第一步中识别基因修饰的生物体，然后在第二步中切除可见的标记。在这里，我们提出了一个一步法来执行精确和快速的内源性基因标记在果蝇中，这使得筛选工程线没有可见的眼睛标记，提供了一个显著优于过去的方法。为了筛选成功的基因标记事件，我们采用基于 PCR 的技术通过分析从中腿的一小段来对单个蝇进行基因分型。通过筛选标准的蝇然后用于产生稳定的股票。在这里，我们详细介绍了 CRISPR 编辑质粒的设计和构建以及工程线的筛选和确认方法。总的来说，该方案大大提高了在果蝇中进行内源基因标记的效率，并能够进行体内细胞过程的研究。
