Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Feb;56(2):10.1002/dvg.23087.
doi: 10.1002/dvg.23087. Epub 2017 Dec 27.

The mito::mKate2 mouse: A far-red fluorescent reporter mouse line for tracking mitochondrial dynamics in vivo

Anthony P Barrasso  1   2 , Xuefei Tong  1 , Ross A Poché  1   3   2
Affiliations

The mito::mKate2 mouse: A far-red fluorescent reporter mouse line for tracking mitochondrial dynamics in vivo

Anthony P Barrasso et al. Genesis. 2018 Feb.

Abstract

Mitochondria are incredibly dynamic organelles that undergo continuous fission and fusion events to control morphology, which profoundly impacts cell physiology including cell cycle progression. This is highlighted by the fact that most major human neurodegenerative diseases are due to specific disruptions in mitochondrial fission or fusion machinery and null alleles of these genes result in embryonic lethality. To gain a better understanding of the pathophysiology of such disorders, tools for the in vivo assessment of mitochondrial dynamics are required. It would be particularly advantageous to simultaneously image mitochondrial fission-fusion coincident with cell cycle progression. To that end, we have generated a new transgenic reporter mouse, called mito::mKate2 that ubiquitously expresses a mitochondria localized far-red mKate2 fluorescent protein. Here we show that mito::mKate2 mice are viable and fertile and that mKate2 fluorescence can be spectrally separated from the previously developed Fucci cell cycle reporters. By crossing mito::mKate2 mice to the ROSA26R-mTmG dual fluorescent Cre reporter line, we also demonstrate the potential utility of mito::mKate2 for genetic mosaic analysis of mitochondrial phenotypes.

Keywords: far-red fluorescence; live imaging; mitochondria.

PubMed Disclaimer

Figures

Figure 1
Figure 1. In vitro validation of the CAG-mito::mKate2 transgene and generation of mice
Map of the CAG-driven mito::mKate2 transgene (A). HeLa cell transiently transfected with the CAG-mito::mKate2 transgene-containing plasmid (B). Emission spectrum fingerprinting of mito::mKate2 fluorescence in transfected HeLa cells (C). Stills from a time-lapse movie of a mito::mKate2 transfected HeLa cell (D). Stills from a movie highlighting a mitochondrial fission event (pseudo-colored in green) occurring in a mito::mKate2 transfected HeLa cell (E). PCR genotyping of 4 recovered CAG-mito::mKate2 transgenic founder mice (F). MTS = mitochondrial targeting signal.
Figure 2
Figure 2. Widespread mitochondrial labeling in the CAG-mito::mKate2 transgenic line
Hind paw of a CAG-mito::mKate2+ transgenic founder progeny showing bright mKate2 fluorescence as compared to a negative littermate (A–B). Expression of mito::mKate2 and resolution of tissue-specific mitochondrial morphologies and distribution in the adult flexor digitorum brevis (FDB) muscle (C), sperm midpiece (D), Purkinje neurons of the cerebellum (E), retina (F), heart (G) and liver (H). GCL=Ganglion Cell Layer, IPL=Inner Plexiform Layer, INL=Inner Nuclear Layer, OPL=Outer Plexiform Layer, ONL=Outer Nuclear Layer, IS=Inner Segments, OS=Outer Segments, CV=Central Vein. White nuclear stain is DAPI. n = 3.
Figure 3
Figure 3. Co-localization of mito::mKate2 fluorescence and mitochondrial-specific markers
HeLa cells transfected with the CAG-mito::mKate2 plasmid and labeled with MitoTracker Green (A). Mito::mKate2 transgenic MEFs co-labeled with MitoTracker Green (B). Mito::mKate2 transgenic retinal cryosections stained with anti-SDH antibodies (C). Red lines demarcate pixels used for co-localization analyses. n = 3. See text for details.
Figure 4
Figure 4. Mito::mKate2 fluorescence can be spectrally separated from the ROSA26R-mTmG dual fluorescent Cre reporter
Postnatal day 0 head cryosections from Pdgfrα-Cre+/tg; ROSA26R-mTmG+/tg; mito::mKate2+/tg mice showing distinct mKate2, GFP and tdTomato fluorescent signals in the choroid plexus (A), osteon (B), and retina (C–D). EC= Ependymal Cells, CC= Choroid Capillaries, HC= Haversian Canal, NBL= Neuroblastic Layer, and GCL= Ganglion Cell Layer. n = 3. See text for details.
Figure 5
Figure 5. Mito::mKate2 fluorescence can be spectrally separated from the Fucci cell cycle reporter
E13.5 embryo cryosections from Fucci-AzG+/tg; Fucci-KO2+/tg; mito::mKate2+/tg mice highlighting distinct fluorescence signals in the mKate2, KO2 and AzG channels throughout the embryo (A). High magnification images of the cerebral cortex (B), heart (C), liver (D) and eye (E). R= Retina and L = Lens. n = 3. See text for details.
Figure 6
Figure 6. Live imaging and structural quantification of mKate2+ mitochondrial dynamics coupled to cell cycle kinetics
Stills from a time-lapse movie of MEFs, derived from Fucci-AzG+/tg; Fucci-KO2+/tg; mito::mKate2+/tg embryos, showing changes in mitochondrial fission and fusion coincident with specific cell cycle stages (A). Connected component analysis (Imaris) of mitochondrial networks, during G1, S and G2 phase (B–D’). The number of individual mKate2+ pixel groups (E) and volume of each group (F) were quantified. As a visual representation of the quantification, individual connected components were randomly color-coded (B’–D’). n = 8 per cell cycle stage. See text for details.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Abe T, Kiyonari H, Shioi G, Inoue K, Nakao K, Aizawa S, Fujimori T. Establishment of conditional reporter mouse lines at ROSA26 locus for live cell imaging. Genesis. 2011;49:579–590. - PubMed
    1. Betsholtz C. Role of platelet-derived growth factors in mouse development. Int J Dev Biol. 1995;39:817–825. - PubMed
    1. Boncompagni S, Rossi AE, Micaroni M, Beznoussenko GV, Polishchuk RS, Dirksen RT, Protasi F. Mitochondria are linked to calcium stores in striated muscle by developmentally regulated tethering structures. Mol Biol Cell. 2009;20:1058–1067. - PMC - PubMed
    1. Breckwoldt MO, Pfister FM, Bradley PM, Marinkovic P, Williams PR, Brill MS, Plomer B, Schmalz A, St Clair DK, Naumann R, Griesbeck O, Schwarzlander M, Godinho L, Bareyre FM, Dick TP, Kerschensteiner M, Misgeld T. Multiparametric optical analysis of mitochondrial redox signals during neuronal physiology and pathology in vivo. Nat Med. 2014;20:555–560. - PubMed
    1. Chan DC. Mitochondria: dynamic organelles in disease, aging, and development. Cell. 2006;125:1241–1252. - PubMed

Publication types

Substances