Heinz Kathrin S, Cardoso M Cristina
Cell Biology and Epigenetics, Technische Universität Darmstadt, 64287, Darmstadt, Germany.
Methods Mol Biol. 2019;2038:199-208. doi: 10.1007/978-1-4939-9674-2_13.
Technical advances in live-cell imaging have made cell biology into a highly dynamic field, allowing the visualization and quantification of complex processes in individual cells and in real time. To follow changes and to specifically manipulate factors potentially involved in processes like DNA replication, transcription or repair, we set up a universal targeting approach, allowing directed manipulation of subcellular structures and molecules therein. This strategy is based on the very strong and specific interaction of GFP and GFP-binding nanobody. We describe in detail how to set up the targeting approach with appropriate controls, as well as how to improve and validate its efficiency and finally provide exemplary applications.
活细胞成像技术的进步使细胞生物学成为一个高度动态的领域，能够对单个细胞中的复杂过程进行可视化和定量分析，并实现实时监测。为了跟踪变化并特异性地操纵可能参与DNA复制、转录或修复等过程的因素，我们建立了一种通用的靶向方法，可对亚细胞结构及其内部的分子进行定向操纵。该策略基于绿色荧光蛋白（GFP）与GFP结合纳米抗体之间非常强且特异的相互作用。我们详细描述了如何设置带有适当对照的靶向方法，以及如何提高和验证其效率，最后提供了示例性应用。
