Improved tools for the Brainbow toolbox


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.


  1. 1

    Tsien, R.Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).

    CAS  Article  Google Scholar 

  2. 2

    Heim, R. & Tsien, R.Y. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr. Biol. 6, 178–182 (1996).

    CAS  Article  Google Scholar 

  3. 3

    Rizzo, M.A., Springer, G.H., Granada, B. & Piston, D.W. An improved cyan fluorescent protein variant useful for FRET. Nat. Biotechnol. 22, 445–449 (2004).

    CAS  Article  Google Scholar 

  4. 4

    Shagin, D.A. et al. GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity. Mol. Biol. Evol. 21, 841–850 (2004).

    CAS  Article  Google Scholar 

  5. 5

    Ai, H.W., Henderson, J.N., Remington, S.J. & Campbell, R.E. Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging. Biochem. J. 400, 531–540 (2006).

    CAS  Article  Google Scholar 

  6. 6

    Subach, O.M. et al. Conversion of red fluorescent protein into a bright blue probe. Chem. Biol. 15, 1116–1124 (2008).

    CAS  Article  Google Scholar 

  7. 7

    Ai, H.W., Shaner, N.C., Cheng, Z., Tsien, R.Y. & Campbell, R.E. Exploration of new chromophore structures leads to the identification of improved blue fluorescent proteins. Biochemistry 46, 5904–5910 (2007).

    CAS  Article  Google Scholar 

  8. 8

    Karasawa, S., Araki, T., Nagai, T., Mizuno, H. & Miyawaki, A. Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer. Biochem. J. 381, 307–312 (2004).

    CAS  Article  Google Scholar 

  9. 9

    Shaner, N.C. et al. Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat. Methods 5, 545–551 (2008).

    CAS  Article  Google Scholar 

  10. 10

    Shaner, N.C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572 (2004).

    CAS  Article  Google Scholar 

  11. 11

    Shcherbo, D. et al. Far-red fluorescent tags for protein imaging in living tissues. Biochem. J. 418, 567–574 (2009).

    CAS  Article  Google Scholar 

  12. 12

    Shcherbo, D. et al. Near-infrared fluorescent proteins. Nat. Methods 7, 827–829 (2010).

    CAS  Article  Google Scholar 

  13. 13

    Shaner, N.C., Steinbach, P.A. & Tsien, R.Y. A guide to choosing fluorescent proteins. Nat. Methods 2, 905–909 (2005).

    CAS  Article  Google Scholar 

  14. 14

    Feng, G. et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41–51 (2000).

    CAS  Article  Google Scholar 

  15. 15

    Livet, J. et al. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 56–62 (2007).

    CAS  Article  Google Scholar 

  16. 16

    Branda, C.S. & Dymecki, S.M. Talking about a revolution: the impact of site-specific recombinases on genetic analyses in mice. Dev. Cell 6, 7–28 (2004).

    CAS  Article  Google Scholar 

  17. 17

    Snippert, H.J. et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143, 134–144 (2010).

    CAS  Article  Google Scholar 

  18. 18

    Red-Horse, K., Ueno, H., Weissman, I.L. & Krasnow, M.A. Coronary arteries form by developmental reprogramming of venous cells. Nature 464, 549–553 (2010).

    CAS  Article  Google Scholar 

  19. 19

    Rinkevich, Y., Lindau, P., Ueno, H., Longaker, M.T. & Weissman, I.L. Germ-layer and lineage-restricted stem/progenitors regenerate the mouse digit tip. Nature 476, 409–413 (2011).

    CAS  Article  Google Scholar 

  20. 20

    Schepers, A.G. et al. Lineage tracing reveals Lgr5+ stem cell activity in mouse intestinal adenomas. Science 337, 730–735 (2012).

    CAS  Article  Google Scholar 

  21. 21

    Tabansky, I. et al. Developmental bias in cleavage-stage mouse blastomeres. Curr. Biol. 23, 21–31 (2013).

    CAS  Article  Google Scholar 

  22. 22

    Gupta, V. & Poss, K.D. Clonally dominant cardiomyocytes direct heart morphogenesis. Nature 484, 479–484 (2012).

    CAS  Article  Google Scholar 

  23. 23

    Pan, Y.A., Livet, J., Sanes, J.R., Lichtman, J.W. & Schier, A.F. Multicolor Brainbow imaging in zebrafish. Cold Spring Harb. Protoc. 2011, pdb.prot5546 (2011).

  24. 24

    Hampel, S. et al. Drosophila Brainbow: a recombinase-based fluorescence labeling technique to subdivide neural expression patterns. Nat. Methods 8, 253–259 (2011).

    CAS  Article  Google Scholar 

  25. 25

    Hadjieconomou, D. et al. Flybow: genetic multicolor cell labeling for neural circuit analysis in Drosophila melanogaster. Nat. Methods 8, 260–266 (2011).

    CAS  Article  Google Scholar 

  26. 26

    Lang, C., Guo, X., Kerschensteiner, M. & Bareyre, F.M. Single collateral reconstructions reveal distinct phases of corticospinal remodeling after spinal cord injury. PLoS ONE 7, e30461 (2012).

    CAS  Article  Google Scholar 

  27. 27

    Badaloni, A. et al. Transgenic mice expressing a dual, CRE-inducible reporter for the analysis of axon guidance and synaptogenesis. Genesis 45, 405–412 (2007).

    CAS  Article  Google Scholar 

  28. 28

    Lichtman, J.W., Livet, J. & Sanes, J.R. A technicolour approach to the connectome. Nat. Rev. Neurosci. 9, 417–422 (2008).

    CAS  Article  Google Scholar 

  29. 29

    Lakso, M. et al. Targeted oncogene activation by site-specific recombination in transgenic mice. Proc. Natl. Acad. Sci. USA 89, 6232–6236 (1992).

    CAS  Article  Google Scholar 

  30. 30

    Paterna, J.C., Moccetti, T., Mura, A., Feldon, J. & Büeler, H. Influence of promoter and WHV post-transcriptional regulatory element on AAV-mediated transgene expression in the rat brain. Gene Ther. 7, 1304–1311 (2000).

    CAS  Article  Google Scholar 

  31. 31

    Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010).

    CAS  Article  Google Scholar 

  32. 32

    Hippenmeyer, S. et al. A developmental switch in the response of DRG neurons to ETS transcription factor signaling. PLoS Biol. 3, e159 (2005).

    Article  Google Scholar 

  33. 33

    Srinivas, S. et al. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev. Biol. 1, 4 (2001).

    CAS  Article  Google Scholar 

  34. 34

    Guo, C., Yang, W. & Lobe, C.G. A Cre recombinase transgene with mosaic, widespread tamoxifen-inducible action. Genesis 32, 8–18 (2002).

    CAS  Article  Google Scholar 

  35. 35

    Zhang, X.M. et al. Highly restricted expression of Cre recombinase in cerebellar Purkinje cells. Genesis 40, 45–51 (2004).

    Article  Google Scholar 

  36. 36

    Rossi, J. et al. Melanocortin-4 receptors expressed by cholinergic neurons regulate energy balance and glucose homeostasis. Cell Metab. 13, 195–204 (2011).

    CAS  Article  Google Scholar 

  37. 37

    Campsall, K.D., Mazerolle, C.J., De Repentingy, Y., Kothary, R. & Wallace, V.A. Characterization of transgene expression and Cre recombinase activity in a panel of Thy-1 promoter-Cre transgenic mice. Dev. Dyn. 224, 135–143 (2002).

    CAS  Article  Google Scholar 

  38. 38

    Bunting, M., Bernstein, K.E., Greer, J.M., Capecchi, M.R. & Thomas, K.R. Targeting genes for self-excision in the germ line. Genes Dev. 13, 1524–1528 (1999).

    CAS  Article  Google Scholar 

  39. 39

    McLeod, M., Craft, S. & Broach, J.R. Identification of the crossover site during FLP-mediated recombination in the Saccharomyces cerevisiae plasmid 2 μm circle. Mol. Cell Biol. 6, 3357–3367 (1986).

    CAS  Article  Google Scholar 

  40. 40

    Schlake, T. & Bode, J. Use of mutated FLP recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci. Biochemistry 33, 12746–12751 (1994).

    CAS  Article  Google Scholar 

  41. 41

    Peroutka, R.J., Elshourbagy, N., Piech, T. & Butt, T.R. Enhanced protein expression in mammalian cells using engineered SUMO fusions: secreted phospholipase A2. Protein Sci. 17, 1586–1595 (2008).

    CAS  Article  Google Scholar 

  42. 42

    Farley, F.W., Soriano, P., Steffen, L.S. & Dymecki, S.M. Widespread recombinase expression using FLPeR (flipper) mice. Genesis 28, 106–110 (2000).

    CAS  Article  Google Scholar 

  43. 43

    Awatramani, R., Soriano, P., Rodriguez, C., Mai, J.J. & Dymecki, S.M. Cryptic boundaries in roof plate and choroid plexus identified by intersectional gene activation. Nat. Genet. 35, 70–75 (2003).

    CAS  Article  Google Scholar 

  44. 44

    Araki, K., Okada, Y., Araki, M. & Yamamura, K. Comparative analysis of right element mutant loxP sites on recombination efficiency in embryonic stem cells. BMC Biotechnol. 10, 29 (2010).

    Article  Google Scholar 

  45. 45

    Entenberg, D. et al. Setup and use of a two-laser multiphoton microscope for multichannel intravital fluorescence imaging. Nat. Protoc. 6, 1500–1520 (2011).

    CAS  Article  Google Scholar 

  46. 46

    Mahou, P. et al. Multicolor two-photon tissue imaging by wavelength mixing. Nat. Methods 9, 815–818 (2012).

    CAS  Article  Google Scholar 

  47. 47

    Wang, K. et al. Three-color femtosecond source for simultaneous excitation of three fluorescent proteins in two-photon fluorescence microscopy. Biomed. Opt. Express 3, 1972–1977 (2012).

    CAS  Article  Google Scholar 

  48. 48

    Conchello, J.A. & Lichtman, J.W. Optical sectioning microscopy. Nat. Methods 2, 920–931 (2005).

    CAS  Article  Google Scholar 

  49. 49

    Ducros, M. et al. Efficient large core fiber-based detection for multi-channel two-photon fluorescence microscopy and spectral unmixing. J. Neurosci. Methods 198, 172–180 (2011).

    CAS  Article  Google Scholar 

  50. 50

    Card, J.P. et al. A dual infection pseudorabies virus conditional reporter approach to identify projections to collateralized neurons in complex neural circuits. PLoS ONE 6, e21141 (2011).

    CAS  Article  Google Scholar 

  51. 51

    Hancock, J.F., Cadwallader, K., Paterson, H. & Marshall, C.J.A. CAAX or a CAAL motif and a second signal are sufficient for plasma membrane targeting of ras proteins. EMBO J. 10, 4033–4039 (1991).

    CAS  Article  Google Scholar 

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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




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)


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).

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