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Nat Protoc:华东理工大学杨弋教授开发新型细胞检测传感器

摘要 : 2016年6月30日,国际学术权威刊物自然出版集团旗下子刊《Nature Protocols》在线发表了华东理工大学药学院、生物反应器工程国家重点实验室特聘杨弋教授的一篇研究论文

 2016年6月30日,国际学术权威刊物自然出版集团旗下子刊《Nature Protocols》在线发表了华东理工大学药学院、生物反应器工程国家重点实验室特聘杨弋教授的一篇研究论文,论文报告称研究人员发明了对NAD+/NADH氧化还原状态高度敏感的一种传感器SoNar,该传感器可用于体内监测细胞的能量代谢。

文章中,研究人员提供了利用高度敏感的、遗传编码的荧光传感器SoNar来成像及监测活细胞与体内NAD+/NADH氧化还原状态的一份详细的实验方案。根据研究人员的描述,这一嵌合SoNar蛋白最初是通过将环状排列的黄色荧光蛋白(cpYFP)插入到来自水生栖热菌的Rex蛋白(T-Rex)的NADH结合结构域中而开发出来的。它通过结合NAD+或NADH,由此诱导影响荧光特性的蛋白质构象改变来发挥功能。

研究人员描述了如何来建立SoNar表达蛋白的步骤,并随后讨论了如何利用这一系统来量化细胞内的氧化还原状态。这种方法敏感、准确、简单,且能够实时报告各种能量代谢信号通路的微小扰动。他们还详细描述了在一项基于酶标仪的检测中应用SoNar对靶向细胞代谢的候选化合物进行了高通量化学筛查,并在小鼠中对表达SoNar的移植瘤进行了体内荧光成像。通常情况下,在活细胞和活体小鼠中SoNar荧光成像的大致时间范围分别为30分钟和60分钟。作者们指出,对于在384孔板中进行高通量化学筛查,整个程序通常需要不超过60分钟的时间,就可以评估出380种化合物对细胞代谢的影响。

原文链接:

In vivo monitoring of cellular energy metabolism using SoNar, a highly responsive sensor for NAD+/NADH redox state

原文摘要:

NADH and its oxidized form NAD+ have a central role in energy metabolism, and their concentrations are often considered to be among the most important readouts of metabolic state. Here, we present a detailed protocol to image and monitor NAD+/NADH redox state in living cells and in vivo using a highly responsive, genetically encoded fluorescent sensor known as Sonar (sensor of NAD(H) redox). The chimeric Sonar protein was initially developed by inserting circularly permuted yellow fluorescent protein (cpYFP) into the NADH-binding domain of Rex protein fromThermus aquaticus (T-Rex). It functions by binding to either NAD+ or NADH, thus inducing protein conformational changes that affect its fluorescent properties. We first describe steps for how to establish SoNar-expressing cells, and then discuss how to use the system to quantify the intracellular redox state. This approach is sensitive, accurate, simple and able to report subtle perturbations of various pathways of energy metabolism in real time. We also detail the application of Sonar to high-throughput chemical screening of candidate compounds targeting cell metabolism in a microplate-reader-based assay, along with in vivo fluorescence imaging of tumor xenografts expressing Sonar in mice. Typically, the approximate time frame for fluorescence imaging of Sonar is 30 min for living cells and 60 min for living mice. For high-throughput chemical screening in a 384-well-plate assay, the whole procedure generally takes no longer than 60 min to assess the effects of 380 compounds on cell metabolism.

来源: Nature Protocols 浏览次数:0

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