Engineering fluorescence and magnetic resonance reporter genes for imaging biological function in hypoxic cells and in vivo

项目摘要 研究生物功能的最有力的方法之一依赖于使用基因编码的 发光蛋白质使细胞生理学可视化。然而，现有的记者基因-原型绿色 荧光蛋白和荧光素酶−有两个主要的局限性。首先，分子氧的氧化是 绿色荧光蛋白、荧光素酶和衍生记者发光机制的核心。第二，光学光子 被不透明的组织分散和吸收，有效地阻止了光线在完好动物体内的穿透。作为一名 结果，基于GFP和荧光素酶的记者在复杂的环境中不能产生光，如缺氧(氧气和lt； 1%)或在完好无损的动物体内。这些缺陷的直接影响是医学研究。低氧 在肿瘤和多重微生物感染的病理生理学中起着中心作用，其后果范围广泛 从耐药性到炎症。为了了解低氧如何在这些情况下重新编程细胞功能， 需要能够在低氧环境中动态研究生物活性的报告基因技术 细胞培养。同样，为了理解肿瘤生物学等过程在体内的重要背景，有 是对与光学不透明动物兼容的报告基因的需求。我们研究计划的目标是 是为了解决这些生物成像领域长期存在的挑战。要做到这一点，我们的研究将继续 新型低氧细胞生物功能无创成像报告基因的研究进展 培养(体外)和活体动物(体内)。我们提出的方法建立在具有特殊性质的蛋白质上 -光感受器，顺磁酶和水通道-并应用分子工程来开发 新的荧光和磁共振成像(MRI)记者。我们的研究计划提出了五个核心 目的：1)设计明亮、多色、不依赖氧的低氧荧光蛋白；2) 开发用于活体成像的灵敏和可多路传输的MRI记录器，3)设计生物响应传感器 基于这些蛋白质来检测细胞代谢物和基因表达，4)将这些传感器应用于研究 缺氧细菌对抗生素的耐受性，以及5)诱导耐特殊治疗的癌细胞 胶质母细胞瘤。这些目标的成功将为研究广谱提供突破性的技术 在生物过程中，缺氧和体内环境提供了重要的病理生理背景。

Project Summary One of the most powerful approaches for studying biological function relies on the use of genetically encoded light-emitting proteins to visualize cell physiology. However, existing reporter genes - the prototypical green fluorescent protein (GFP) and luciferase − have two major limitations. First, oxidation by molecular oxygen is central to the mechanism by which GFP, luciferase, and derivative reporters emit light. Second, optical photons are scattered and absorbed by opaque tissue, which effectively blocks light penetration in intact animals. As a result, GFP and luciferase based reporters fail to produce light in complex settings such as hypoxia (oxygen < 1%) or deep inside intact animals. An immediate impact of these shortcomings is on medical research. Hypoxia plays a central role in the pathophysiology of tumors and polymicrobial infections with consequences ranging from drug resistance to inflammation. To understand how hypoxia reprograms cell function in these contexts, there is a need for reporter gene technologies that allow biological activity to be dynamically studied in hypoxic cell cultures. Likewise, to understand processes such as tumor biology in their important in vivo context, there is a need for reporter genes that are compatible with optically opaque animals. The goal of our research program is to address these long-standing challenges in biological imaging. To do so, our research will pursue the development of new classes of reporter genes for noninvasive imaging of biological function in hypoxic cell cultures (in vitro) and in live animals (in vivo). Our proposed approach builds on proteins with special properties – photoreceptors, paramagnetic enzymes, and water channels – and applies molecular engineering to develop new reporters for fluorescence and magnetic resonance imaging (MRI). Our research program proposes five core objectives: 1) engineering bright, multi-colored, oxygen-independent fluorescent proteins for hypoxia, 2) developing sensitive and multiplexable MRI reporters for in vivo imaging, 3) designing bioresponsive sensors based on these proteins to detect cell metabolites and gene expression, 4) applying these sensors to study antibiotic tolerance in hypoxic bacteria, and 5) induction of specialized treatment resistant cancer cells in glioblastoma tumors. Success in these goals will provide a breakthrough technique for studying a broad spectrum of biological processes where hypoxia and in vivo milieu provide important pathophysiological contexts.