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Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system

Nature volume 450, pages 5662 (01 November 2007) | Download Citation

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Abstract

Detailed analysis of neuronal network architecture requires the development of new methods. Here we present strategies to visualize synaptic circuits by genetically labelling neurons with multiple, distinct colours. In Brainbow transgenes, Cre/lox recombination is used to create a stochastic choice of expression between three or more fluorescent proteins (XFPs). Integration of tandem Brainbow copies in transgenic mice yielded combinatorial XFP expression, and thus many colours, thereby providing a way to distinguish adjacent neurons and visualize other cellular interactions. As a demonstration, we reconstructed hundreds of neighbouring axons and multiple synaptic contacts in one small volume of a cerebellar lobe exhibiting approximately 90 colours. The expression in some lines also allowed us to map glial territories and follow glial cells and neurons over time in vivo. The ability of the Brainbow system to label uniquely many individual cells within a population may facilitate the analysis of neuronal circuitry on a large scale.

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Acknowledgements

We thank members of the Lichtman and Sanes laboratories for discussions; H. Nishimune for insights; A. Mendelsohn and A. Bruskin for cell culture and PCR; S.-H. Shieu and D. Mou for imaging software development; S. Haddad, D. Pelusi, D. Malkowski, K. Mahoney and the Harvard MCB BRI facility for animal care and genotyping; M. Wallace, the WUSTL Mouse Genetic Core and the MCB Genome Manipulation Facility for pronuclei injections; and R. Hellmiss for help with graphics. We thank J. C. Fiala for assistance with Reconstruct; M. L. Nonet, J. S. Mumm and R. O. Wong for advice and reagents; D. W. Piston for mCerulean; R. Y. Tsien for tdimer2 and mCherry; and A. Miyawaki for Kusabira. This work was supported by the James S. McDonnell foundation and grants from NIH.

Author Contributions J.L., J.R.S. and J.W.L. conceived the Brainbow strategies. J.R.S. and J.W.L. supervised the project. J.L. built initial constructs and validated them in vitro and in vivo. T.A.W. performed all cerebellar axonal tracing and colour profile analysis with programs developed with J. Lu. H.K. performed all live imaging experiments. R.W.D. generated Brainbow-1.0 lines expressing cytoplasmic XFPs, and R.A.B. generated Brainbow-1.1 constructs and lines. J.L., T.A.W. and R.W.D. screened mouse lines.

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Affiliations

  1. Department of Molecular and Cellular Biology and Center for Brain Science, Harvard University, Cambridge, Massachusetts 02138, USA

    • Jean Livet
    • , Tamily A. Weissman
    • , Hyuno Kang
    • , Ryan W. Draft
    • , Ju Lu
    • , Robyn A. Bennis
    • , Joshua R. Sanes
    •  & Jeff W. Lichtman

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The authors declare no competing financial interests.

Corresponding author

Correspondence to Jeff W. Lichtman.

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    Supplementary Information

    The file contains Supplementary Figures 1-6 with Legends and Supplementary Tables 1-2.

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    Supplementary Movie 1

    The file contains Supplementary Movie 1 which shows rotation of the cerebellar reconstruction.

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https://doi.org/10.1038/nature06293

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