Versatile genetic paintbrushes: Brainbow technologies.
Bottom Line: While being continuously refined, Brainbow technologies have thus found a firm place in the genetic toolboxes of developmental and neurobiologists.For further resources related to this article, please visit the WIREs website.The authors have declared no conflicts of interest for this article.
Affiliation: MRC National Institute for Medical Research, Division of Molecular Neurobiology, London, UK.Show MeSH
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Mentions: dBrainbow3 is modeled on the Brainbow-1 strategy and uses Cre-mediated recombination of heterospecific lox sites (Figure 3(a)). A transcriptional stop cassette precedes the series of three FP-encoding sequences to ensure that cells are solely labeled upon Cre expression. Moreover, each FP is tagged with a different epitope (V5, HA, and myc), which can be detected by immunohistochemistry. This helps to boost labeling intensities when endogenous fluorescence signals are inherently low or quenched during fixation of tissues. By contrast, Flybow transgenes4 are based on the Brainbow-2 strategy (Figure 3(b)). To bypass the limitations of Cre in flies, Flybow uses the mFLP5-mFRT71 system77 as an orthogonal tool that can be combined with the canoncial FLP-FRT system. mFLP5 is controlled by the heat-shock promoter. Transient exposure to heat induces the expression of mFLP5, which mediates inversions and excisions of cassettes flanked by mFRT71 sites. Moreover, to facilitate complete labeling of neurites, all FPs are membrane-tethered.43,36 Similar to mouse Brainbow-2.0 and -2.1 transgenes, Flybow-1.0 and -1.1 constructs consist of one and two cassettes, respectively. In Flybow-2.0, an additional transcriptional stop cassette flanked by FRT sites in the same orientation precedes the invertible cassettes to eliminate default marker expression. The stop cassette is excised after induction of the canonical FLP recombinase. Transient FLP expression facilitates both sparse labeling and increases the color diversity because all four FPs can be used for tracing. Because Flybow-2.0 relies on both FLP and mFLP5, it additionally can serve as an intersectional tool to refine expression, when FLP expression is controlled by a different cell-specific enhancer. The initial set of Flybow constructs uses an epitope-tagged cyan FP mCerulean variant,31 which requires immunodetection because of its weak native emission in flies. To bypass the need for immunolabeling and to enable live imaging of endogenous fluorescence signals in all four channels, in a second set of transgenes (Flybow-1.0B, 1.1B and 2.0B)5 cd8-tethered mCerulean-V5 was replaced by the brighter myr-palm anchored mTurquoise.32
Affiliation: MRC National Institute for Medical Research, Division of Molecular Neurobiology, London, UK.