Improved tools for the Brainbow toolbox

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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Figure 1: Brainbow 3 transgenic mice.
Figure 2: Improved visualization of neurons in Brainbow 3 mice.
Figure 3: Autobow.
Figure 4: Flpbow.
Figure 5: Brainbow AAV.
Figure 6: Processing a Brainbow image.

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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.

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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.

Corresponding author

Correspondence to Joshua R Sanes.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11 and Supplementary Tables 1 and 2 (PDF 2371 kb)

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. (AVI 6653 kb)

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. (AVI 5501 kb)

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. (AVI 6597 kb)

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Cai, D., Cohen, K., Luo, T. et al. Improved tools for the Brainbow toolbox. Nat Methods 10, 540–547 (2013). https://doi.org/10.1038/nmeth.2450

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