Hu Huan, Yang Xiaojing, Tang Chao
School of Life Sciences, Peking University, Beijing, China.
Center for Quantitative Biology, Peking University, Beijing, China.
Curr Protoc Cell Biol. 2019 Mar;82(1):e78. doi: 10.1002/cpcb.78. Epub 2018 Oct 30.
Tracking the dynamics of genomic loci is essential for understanding a variety of cellular processes. However, earlier methods have all suffered from a low signal-to-background ratio (SBR), mainly caused by the background fluorescence from diffuse full-length fluorescent proteins in the nucleus. We have developed a novel method (BiFC-TALE) for labeling and tracking genomic loci in live mammalian cells, combining bimolecular fluorescence complementation (BiFC) and transcription activator-like effector (TALE) technologies. Since only the sequences-targeted BiFC fragments can be pulled together by TALE modules to recombine intact fluorescent proteins, the background fluorescence in the living nucleus can be largely reduced, which significantly improves SBR. Using telomere and centromere labeling as examples, this unit describes in detail the design and implementation of BiFC-TALE system. © 2018 by John Wiley & Sons, Inc.
追踪基因组位点的动态变化对于理解各种细胞过程至关重要。然而，早期的方法都存在低信噪比（SBR）的问题，这主要是由细胞核中弥漫的全长荧光蛋白产生的背景荧光所致。我们开发了一种用于在活的哺乳动物细胞中标记和追踪基因组位点的新方法（双分子荧光互补-转录激活样效应因子，BiFC-TALE），该方法结合了双分子荧光互补（BiFC）和转录激活样效应因子（TALE）技术。由于只有靶向序列的BiFC片段能够被TALE模块拉到一起重新组合成完整的荧光蛋白，因此活细胞核中的背景荧光能够大幅降低，这显著提高了信噪比。以端粒和着丝粒标记为例，本单元详细描述了BiFC-TALE系统的设计与实施。© 2018约翰威立国际出版公司。
