基本 helix-loop-helix (bHLH ) 的成员基因家庭在脊椎动物神经发生起重要作用。在这研究,共焦的基于显微镜学的荧光回声精力转移(烦恼) 被用来在各种各样的生理的条件下面监视 bHLH 蛋白质蛋白质相互作用。织物特定的 bHLH 使活跃之物, NeuroD1, Mash1, Neurogenin1 (Ngn1 ) , Neurogenin2 (Ngn2 ) ,和无所不在的表示 E47 蛋白质与提高的黄荧光蛋白质(EYFP ) 被标注并且分别地提高了青色荧光蛋白质(ECFP ) 。潜水艇 bHLH 熔化蛋白质的细胞的本地化和活动性在 HEK293 房间被检验。由短暂 transfection 并且在 ovo electroporation,四织物特定的 bHLH 使活跃之物和 E47 蛋白质在 HEK293 房间和发展中的小鸡胚胎是过去表示的神经试管。与漂白的领受人相片方法,烦恼能在 transfected 的原子核在这些 bHLH 蛋白质对之间被检测房间和发展中的神经试管。Mash1/E47 和 Ngn2/E47 烦恼对更高出现烦恼效率在中间并且小鸡胚胎的侧面的一半神经试管分别地。它建议这些 bHLH 蛋白质对在特定的区域与他们的下游的目标基因的规章的元素形成了功能的 DNA 蛋白质建筑群。这个工作将帮助一个在 vivo 理解 bHLH 因素的行为。
Members of the basic helix-loop-helix (bHLH) gene family play important roles in vertebrate neurogenesis. In this study, confocal microscopy-based fluorescence resonance energy transfer (FRET) is used to monitor bHLH protein-protein interactions under various physiological conditions. Tissue-specific bHLH activators, NeuroD 1, Mash 1, Neurogenin 1 (Ngn 1), Neurogenin2 (Ngn2), and ubiquitous expressed E47 protein are tagged with enhanced yellow fluorescence protein (EYFP) and enhanced cyan fluorescence protein (ECFP), respectively. The subcellular localization and mobility ofbHLH fusion proteins are examined in HEK293 cells. By transient transfection and in ovo electroporation, four pairs of tissue-specific bHLH activators and E47 protein are over-expressed in HEK293 cells and developing chick embryo neural tube. With the acceptor photobleaching method, FRET could be detected between these bHLH protein pairs in the nuclei of transfected cells and developing neural tubes. Mashl/E47 and Ngn2/E47 FRET pairs show higher FRET efficiencies in the medial and the lateral half of chick embryo neural tube, respectively. It suggests that these bHLH protein pairs formed functional DNA-protein complexes with regulatory elements of their downstream target genes in the specific regions. This work will help one understand the behaviours of bHLH factors in vivo.