Trinh Le A, Chong-Morrison Vanessa, Gavriouchkina Daria, Hochgreb-Hägele Tatiana, Senanayake Upeka, Fraser Scott E, Sauka-Spengler Tatjana
Molecular and Computational Biology, University of Southern California, Los Angeles, CA 90089, USA.
Radcliffe Department of Medicine, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK.
Cell Rep. 2017 Apr 11;19(2):425-440. doi: 10.1016/j.celrep.2017.03.045.
Interrogation of gene regulatory circuits in complex organisms requires precise tools for the selection of individual cell types and robust methods for biochemical profiling of target proteins. We have developed a versatile, tissue-specific binary in vivo biotinylation system in zebrafish termed biotagging that uses genetically encoded components to biotinylate target proteins, enabling in-depth genome-wide analyses of their molecular interactions. Using tissue-specific drivers and cell-compartment-specific effector lines, we demonstrate the specificity of the biotagging toolkit at the biochemical, cellular, and transcriptional levels. We use biotagging to characterize the in vivo transcriptional landscape of migratory neural crest and myocardial cells in different cellular compartments (ribosomes and nucleus). These analyses reveal a comprehensive network of coding and non-coding RNAs and cis-regulatory modules, demonstrating that tissue-specific identity is embedded in the nuclear transcriptomes. By eliminating background inherent to complex embryonic environments, biotagging allows analyses of molecular interactions at high resolution.
在复杂生物体中研究基因调控回路需要精确的工具来选择单个细胞类型，以及可靠的方法对目标蛋白质进行生化分析。我们在斑马鱼中开发了一种通用的、组织特异性的体内生物素化系统，称为生物标记，该系统使用基因编码组件对目标蛋白质进行生物素化，从而能够对其分子相互作用进行全基因组深度分析。利用组织特异性驱动因子和细胞区室特异性效应子系，我们在生化、细胞和转录水平上证明了生物标记工具包的特异性。我们使用生物标记来表征不同细胞区室（核糖体和细胞核）中迁移神经嵴和心肌细胞的体内转录图谱。这些分析揭示了一个由编码和非编码RNA以及顺式调控模块组成的综合网络，表明组织特异性特征嵌入在核转录组中。通过消除复杂胚胎环境固有的背景，生物标记允许以高分辨率分析分子相互作用。
