School of Life Sciences, Peking University, Beijing 100871, China.
Center for Quantitative Biology, Peking University, Beijing 100871, China.
Sci Rep. 2017 Jan 11;7:40192. doi: 10.1038/srep40192.
Tracking the dynamics of genomic loci is important for understanding the mechanisms of fundamental intracellular processes. However, fluorescent labeling and imaging of such loci in live cells have been challenging. One of the major reasons is the low signal-to-background ratio (SBR) of images mainly caused by the background fluorescence from diffuse full-length fluorescent proteins (FPs) in the living nucleus, hampering the application of live cell genomic labeling methods. Here, combining bimolecular fluorescence complementation (BiFC) and transcription activator-like effector (TALE) technologies, we developed a novel method for labeling genomic loci (BiFC-TALE), which largely reduces the background fluorescence level. Using BiFC-TALE, we demonstrated a significantly improved SBR by imaging telomeres and centromeres in living cells in comparison with the methods using full-length FP.
跟踪基因组基因座的动态对于理解基本细胞内过程的机制很重要。然而，在活细胞中对这些基因座进行荧光标记和成像一直具有挑战性。主要原因之一是图像的信噪比（SBR）低，主要是由于活核中弥漫性全长荧光蛋白（FP）的背景荧光引起的，这阻碍了活细胞基因组标记方法的应用。在这里，我们结合双分子荧光互补（BiFC）和转录激活子样效应物（TALE）技术，开发了一种标记基因组基因座的新方法（BiFC-TALE），该方法大大降低了背景荧光水平。使用 BiFC-TALE，与使用全长 FP 的方法相比，我们通过在活细胞中成像端粒和着丝粒，证明了 SBR 有了显著提高。
