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 发光蛋白质来可视化细胞生理学。然而，现有的报告基因-原型绿色 荧光蛋白（GFP）和荧光素酶-有两个主要的局限性。首先，通过分子氧的氧化， 对GFP、荧光素酶和衍生报告物发光的机制至关重要。第二，光子 被不透明的组织散射和吸收，这有效地阻止了光穿透完整的动物。作为 结果，基于GFP和荧光素酶的报告基因在复杂环境中不能产生光，例如缺氧（氧< 1%）或深入完整的动物体内。这些缺陷的直接影响是医学研究。缺氧 在肿瘤和多种微生物感染的病理生理学中起着核心作用， 从抗药性到炎症为了了解缺氧如何在这些情况下重新编程细胞功能， 需要报道基因技术，其允许在低氧环境中动态研究生物活性， 细胞培养同样，为了理解诸如肿瘤生物学等重要的体内过程， 需要与光学不透明动物相容的报告基因。我们研究项目的目标 是解决生物成像领域长期存在的挑战。为此，我们的研究将追踪 开发新类型的报告基因用于缺氧细胞中生物功能的非侵入性成像 培养物（体外）和活动物（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，顺磁性酶和水通道-并应用分子工程来开发 荧光和磁共振成像（MRI）的新报告者。我们的研究计划提出了五个核心 目的：1）工程化用于缺氧的明亮、多色、不依赖氧的荧光蛋白，2） 开发用于体内成像的灵敏和可复用的MRI报告子，3）设计生物响应传感器 基于这些蛋白质检测细胞代谢产物和基因表达，4）将这些传感器应用于研究 低氧细菌中的抗生素耐受性，和5）在低氧细菌中诱导专门的治疗抗性癌细胞， 胶质母细胞瘤这些目标的成功将为研究广谱 缺氧和体内环境提供重要的病理生理学背景的生物过程。

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.