文献类型：期刊文献

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

作者：Zhao, Yuzheng[1,2,3,4];Wang, Aoxue[1,2,4];Zou, Yejun[1,2,4];Su, Ni[1,2,4];Loscalzo, Joseph[5];Yang, Yi[1,2,3,4]

机构：[1]East China Univ Sci & Technol, Shanghai Collaborat Innovat Ctr Biomfg Technol, State Key Lab Bioreactor Engn, Synthet Biol & Biotechnol Lab, Shanghai, Peoples R China;[2]East China Univ Sci & Technol, CAS Ctr Excellence Brain Sci, Optogenet & Mol Imaging Interdisciplinary Res Ctr, Shanghai, Peoples R China;[3]East China Univ Sci & Technol, Collaborat Innovat Ctr Genet & Dev, Shanghai, Peoples R China;[4]East China Univ Sci & Technol, Sch Pharm, Shanghai Key Lab New Drug Design, Shanghai, Peoples R China;[5]Harvard Med Sch, Brigham & Womens Hosp, Dept Med, Boston, MA USA

年份：2016

卷号：11

期号：8

起止页码：1345

外文期刊名：NATURE PROTOCOLS

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000379814300003)】；

基金：We thank G. Yellen (Harvard Medical School, Boston, MA) for the Peredox vector; M. Chen, S. Lan, S. Han, Y. Ci, R. Yang, L. Yue and Y. Wang for technical assistance; and J. Zou for editing the manuscript. This research was supported by the 973 Program (2013CB531200 to Y.Y.), NSFC (91313301, 31225008, 31071260, 31170815, 31470833 and 91013012 to Y.Y.), the Specialized Research Fund for the Doctoral Program of Higher Education (20100074110010 to Y.Y.), the Shanghai Science and Technology Commission (12JC1402900 and 11DZ2260600 to Y.Y., and 15YF1402600 to Y. Zhao), the Dawn Program of the Shanghai Education Commission (11SG31 to Y.Y.), the Lift Engineering for Young Talent of China Association for Science and Technology (to Y. Zhao), the State Key Laboratory of Bioreactor Engineering (to Y.Y.), the 111 Project (B07023 to Y.Y.), the Fundamental Research Funds for the Central Universities (to Y.Y. and Y. Zhao) and the National Institutes of Health (grants HL061795, HL048743, HL108630 and GM107618 to J.L.).

语种：英文

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 from Thermus 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.