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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Bioluminescence resonance energy transfer (BRET) imaging of protein-protein interactions within deep tissues of living subjects
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Bioluminescence resonance energy transfer (BRET) imaging of protein-protein interactions within deep tissues of living subjects

机译：生物发光共振能量转移（BRET）成像，用于研究对象深层组织内的蛋白质相互作用

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摘要

Identifying protein-protein interactions (PPIs) is essential for understanding various disease mechanisms and developing new therapeutic approaches. Current methods for assaying cellular in-termolecular interactions are mainly used for cells in culture and have limited use for the noninvasive assessment of small animal disease models. Here, we describe red light-emitting reporter systems based on bioluminescence resonance energy transfer (BRET) that allow for assaying PPIs both in cell culture and deep tissues of small animals. These BRET systems consist of the recently developed Renilla reniformis luciferase (RLuc) variants RLuc8 and RLuc8.6, used as BRET donors, combined with two red fluorescent proteins, TagRFP and TurboFP635, as BRET acceptors. In addition to the native coelenterazine luciferase substrate, we used the synthetic derivative coelenterazine-v, which further red-shifts the emission maxima of Renilla luciferases by 35 nm. We show the use of these BRET systems for ratiometric imaging of both cells in culture and deep-tissue small animal tumor models and validate their applicability for studying PPIs in mice in the context of rapamycin-induced FK506 binding protein 12 (FKBP12)-FKBP12 rapamycin binding domain (FRB) association. These red light-emitting BRET systems have great potential for investigating PPIs in the context of drug screening and target validation applications.
机译：识别蛋白质-蛋白质相互作用（PPI）对于理解各种疾病机制和开发新的治疗方法至关重要。目前用于测定细胞内分子间相互作用的方法主要用于培养中的细胞，并且在小动物疾病模型的非侵入性评估中用途有限。在这里，我们描述了基于生物发光共振能量转移（BRET）的红色发光报告系统，该系统可用于分析小动物的细胞培养和深层组织中的PPI。这些BRET系统由用作BRET供体的最近开发的肾性肾病肾变荧光素酶（RLuc）变体RLuc8和RLuc8.6与两个红色荧光蛋白TagRFP和TurboFP635组合作为BRET受体组成。除了天然的腔肠素荧光素酶底物，我们还使用了合成衍生物腔肠素-v，这进一步使海肾荧光素酶的发射最大值红移了35 nm。我们展示了使用这些BRET系统对培养和深层组织小动物肿瘤模型中的细胞进行比例成像，并验证了它们在雷帕霉素诱导的FK506结合蛋白12（FKBP12）-FKBP12雷帕霉素的背景下研究小鼠PPI的适用性绑定域（FRB）关联。这些发红光的BRET系统在药物筛选和靶标验证应用中具有研究PPI的巨大潜力。

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    Anca Dragulescu-Andrasi; Carmel T. Chan; Abhijit De; Tarik F. Massoud; Sanjiv S. Gambhir;

  • 作者单位

    Molecular Imaging Program at Stanford, Bio-X Program and Department of Radiology, Stanford University School of Medicine, Stanford, CA 94305;

    Molecular Imaging Program at Stanford, Bio-X Program and Department of Radiology, Stanford University School of Medicine, Stanford, CA 94305;

    The Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Kharghar, Navi Mumbai 410210, India;

    Molecular Imaging Program at Stanford, Bio-X Program and Department of Radiology, Stanford University School of Medicine, Stanford, CA 94305 Department of Radiology and Department of Oncology, University of Cambridge School of Clinical Medicine, Cambridge CB2 2QQ, United Kingdom;

    Molecular Imaging Program at Stanford, Bio-X Program and Department of Radiology, Stanford University School of Medicine, Stanford, CA 94305 Department of Bioengineering and Department of Materials Science and Engineering, Stanford University School of Medicine, Stanford, CA 94305;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    optical imaging; reporter gene;

    机译：光学成像报告基因;

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