利用活细胞单分子追踪技术定量描述表观遗传调控因子的全球结合和靶标搜索动态。

University of Colorado Denver Chemistry Department, 1150 12 St., Denver, CO 80204, USA.

STAR Protoc. 2021 Nov 16;2(4):100959. doi: 10.1016/j.xpro.2021.100959. eCollection 2021 Dec 17.

This protocol provides instructions to track the global dynamics of single epigenetic regulatory factors in live cells. We describe an approach to generate cell lines that stably express HaloTag-fused proteins. We then use live-cell single-molecule tracking to obtain kinetic populations and residence times. The kinetic parameters obtained can be used to determine important aspects of transcriptional regulation such as target-search time, 3D free diffusion time, and number of non-specific sites sampled before reaching a specific site and compare behaviors across different nuclear environments. For complete details on the use and execution of this protocol, please refer to Kent et al. (2020).

本方案提供了在活细胞中追踪单个表观遗传调控因子的全球动态的说明。我们描述了一种生成稳定表达 HaloTag 融合蛋白的细胞系的方法。然后，我们使用活细胞单分子追踪来获得动力学群体和停留时间。获得的动力学参数可用于确定转录调控的重要方面，例如目标搜索时间、3D 自由扩散时间以及在到达特定位置之前在非特异性位置上的采样次数，并比较不同核环境下的行为。有关此方案的使用和执行的完整详细信息，请参阅 Kent 等人。(2020)。