Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (10): 97-105.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1607

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Mito-OS-Timer:A Targeted Fluorescent Stopwatch for Monitoring Mitochondrial Oxidative Stress

ZHANG Xiao-ni(), WENG Yi-chun, FAN Yi-hao, WANG Xiao-juan, ZHAO Jia-yu, ZHANG Yun-long()   

  1. College of Chemistry,Chemical Engineering and Biotechnology,Donghua University,Shanghai 201620
  • Received:2021-12-30 Online:2022-10-26 Published:2022-11-11
  • Contact: ZHANG Yun-long E-mail:xnn18838917069@163.com;zhyl@dhu.edu.cn

Abstract:

Mitochondrial oxidative stress(Mito-OS)is a kind of imbalance between reactive oxygen species production and antioxidant system in mitochondria. And oxidative stress induced by reactive oxygen species(ROS)is considered as important factors for aging and disease. At present,the evaluation methods of oxidative stress in cells or animal models are mainly based on cell morphology,animal phenotype or production of characteristic metabolites,et al. It is unable to monitor dynamic changes in real time. This study established a mitochondrial oxidative stress fluorescent protein monitoring system,named Mito-OS-Timer,which can monitor the dynamic changes of mitochondrial oxidative stress in real time. Based on the mechanism that dsRed1-E5 red/green fluorescence conversion rate was positively correlated with oxygen concentration change,dsRed1-E5 gene was fused with ATP5PB fragment of ATP synthase subunit located in mitochondrial inner membrane. Then,we established a recombinant plasmid pMito-OS-Timer and stably expressed cell line HEK293T. After being treated with 0-300 μmol/L H2O2 and 0-5 μmol/L rotenone,the results showed that there was a significantly positive correlation between the rate of red-green fluorescence transformation and the degree of mitochondrial oxidative stress. In addition,Mito-OS-Timer was used to detect the enhancement of oxidative stress induced by folliculin(FLCN)gene silencing by pLVX-shFLCN. This system provides a new visualization method for studying mitochondrial oxidative stress.

Key words: mitochondrial oxidative stress, fluorescent protein timer, Mito-OS-Timer, tumor suppressor factor, FLCN gene