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Improved tools for the Brainbow toolbox

Nature Methods volume 10, pages 540547 (2013) | Download Citation

Abstract

In the transgenic multicolor labeling strategy called 'Brainbow', Cre-loxP recombination is used to create a stochastic choice of expression among fluorescent proteins, resulting in the indelible marking of mouse neurons with multiple distinct colors. This method has been adapted to non-neuronal cells in mice and to neurons in fish and flies, but its full potential has yet to be realized in the mouse brain. Here we present several lines of mice that overcome limitations of the initial lines, and we report an adaptation of the method for use in adeno-associated viral vectors. We also provide technical advice about how best to image Brainbow-expressing tissue.

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Acknowledgements

This work was supported by grants from the US National Institutes of Health (5U24NS063931) and the Gatsby Charitable Foundation and by Collaborative Innovation Award no. 43667 from the Howard Hughes Medical Institute. We thank S. Haddad for assistance with mouse colony maintenance; X. Duan, L. Bogart and J. Lefebvre for testing Brainbow mice and AAVs; R.W. Draft for valuable discussions and advice; R.Y. Tsien (University of California, San Diego) for mOrange2 and TagRFPt; and D.M. Chudakov (Institute of Bioorganic Chemistry of the Russian Academy of Sciences) for TagBFP, PhiYFP, mKate2 and eqFP650.

Author information

Affiliations

  1. Center for Brain Science, Harvard University, Cambridge, Massachusetts, USA.

    • Dawen Cai
    • , Kimberly B Cohen
    • , Tuanlian Luo
    • , Jeff W Lichtman
    •  & Joshua R Sanes
  2. Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.

    • Dawen Cai
    • , Kimberly B Cohen
    • , Tuanlian Luo
    • , Jeff W Lichtman
    •  & Joshua R Sanes

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Contributions

D.C., K.B.C. and T.L. performed experiments. D.C., J.W.L. and J.R.S. designed experiments, interpreted results and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Joshua R Sanes.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–11 and Supplementary Tables 1 and 2

Videos

  1. 1.

    Motor axons and neuromuscular junctions in extraocular muscle

    The video shows confocal z–cross-section images and three-dimensional reconstructions on the left and right, respectively, for a Brainbow 3.0 (line D) Islet-Cre mouse with antibody amplification. EGFP shown in blue, mOrange2 in green and mKate2 in red.

  2. 2.

    Cerebellum

    The video shows confocal z–cross-section images for a Brainbow 3.1 (line 3) L7-Cre mouse with antibody amplification. EGFP shown in blue, mOrange2 in green and mKate2 in red.

  3. 3.

    Parvalbumin-positive interneurons in cerebral cortex

    The video shows confocal z cross-sections and three-dimensional reconstructions on the left and right, respectively. Cortex was labeled by Brainbow AAVs injected into a parvalbumin-Cre mouse, and sections were immunostained. mTFP and EYFP are shown green, TagBFP in blue and mCherry in red.

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DOI

https://doi.org/10.1038/nmeth.2450

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