Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

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.

References

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

  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).

    Article  CAS  Google Scholar 

Download references

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

Authors and Affiliations

Authors

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.

Corresponding author

Correspondence to Joshua R Sanes.

Ethics declarations

Competing interests

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)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

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

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing