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Related Concept Videos

Reporter Genes02:11

Reporter Genes

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Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
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Related Experiment Video

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Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors
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A high-performance genetically encoded fluorescent biosensor for imaging physiological peroxynitrite.

Zhijie Chen1, Shen Zhang2, Xinyu Li3

  • 1Department of Chemistry, University of California, Riverside, Riverside, CA 92521, USA.

Cell Chemical Biology
|February 13, 2021
PubMed
Summary

Researchers developed pnGFP-Ultra, a novel biosensor for detecting peroxynitrite (a reactive nitrogen species) in live cells. This tool offers sensitive and selective imaging, advancing the study of its role in diseases.

Keywords:
biosensorfluorescence microscopyfluorescent probesgenetic code expansionlive-cell imagingnoncanonical amino acidsperoxynitriteperoxynitrite productionprotein engineeringreactive nitrogen species

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Reactive nitrogen species (RNS), such as peroxynitrite, are crucial in cellular signaling, stress responses, and disease pathogenesis.
  • Accurate detection of peroxynitrite is vital for understanding its biological roles and associated pathologies.
  • Existing molecular tools for peroxynitrite detection have limitations in selectivity, sensitivity, or noninvasive application.

Purpose of the Study:

  • To develop a high-performance, genetically encodable biosensor for sensitive and selective imaging of peroxynitrite in live cells.
  • To engineer an efficient system for expressing noncanonical amino acid (ncAA)-containing proteins in mammalian cells.
  • To validate the utility of the novel biosensor in relevant biological contexts.

Main Methods:

  • Development of pnGFP-Ultra, a reaction-based biosensor utilizing a p-boronophenylalanine-modified chromophore.
  • Engineering of a mammalian expression system for noncanonical amino acids (ncAAs) to enable biosensor production.
  • Live-cell imaging experiments to assess peroxynitrite detection in activated macrophages and primary glial cells.

Main Results:

  • pnGFP-Ultra demonstrated a significant ~110-fold fluorescence turn-on response specifically to peroxynitrite.
  • The biosensor exhibited minimal cross-reactivity with other reactive oxygen and nitrogen species.
  • Robust detection of peroxynitrite production was achieved in activated immune cells, including macrophages and glial cells.

Conclusions:

  • pnGFP-Ultra is a powerful and versatile tool for the selective and sensitive detection of peroxynitrite in live cells.
  • The developed ncAA expression system broadens the applicability of genetically encoded biosensors in mammalian systems.
  • This biosensor addresses a critical technical gap, enhancing the study of RNS biology and its implications in human diseases.