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A novel Mito‐Timer reporter gene for measurement of mitochondrial quantity and quality in vivo
Author(s) -
Laker Rhianna Che,
Xu Peng,
Ryall Karen,
Sujkowski Alyson,
Zhang Mei,
Adler Paul,
Wessells Robert,
Saucerman Jeff,
Yan Zhen
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.1209.5
Subject(s) - green fluorescent protein , mitochondrion , timer , in vivo , reporter gene , microbiology and biotechnology , biology , skeletal muscle , gene , mitochondrial disease , somatic cell , mitochondrial dna , biochemistry , genetics , anatomy , gene expression , computer science , computer hardware , microcontroller
Mitochondrial dysfunction plays an important role in many diseases, but there is no satisfactory method to assess mitochondrial quantity and quality in vivo. Here we engineered a novel pMito‐Timer reporter gene from the existing pTimer‐1 reporter gene. pMito‐Timer encodes a mitochondria‐targeted green fluorescent protein (GFP) when newly synthesized, which shifts irreversibly to red fluorescence (DsRed) when oxidized. Confocal microscopy confirmed that Mito‐Timer targets to mitochondria in cultured mouse myoblasts, Drosophila heart and indirect flight muscles, and adult mouse skeletal muscle. Ratiometric algorithm showed that conditions that cause mitochondrial stress lead to a significant fluorescence shift toward DsRed with a significant increase of DsRed puncta. Somatic gene transfer in adult skeletal muscle in mice showed that high‐fat diet induces the same changes, which could be completely ameliorated by exercise training. Exercise training also results in increases in mitochondrial volume that could be quantified. Therefore, pMito‐Timer can be used to report mitochondrial health under both normal and pathological conditions and will provide a valuable tool to multiple disciplines investigating the role of the mitochondria in disease.