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Versatile genetic paintbrushes: Brainbow technologies.

Richier B, Salecker I - Wiley Interdiscip Rev Dev Biol (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: MRC National Institute for Medical Research, Division of Molecular Neurobiology, London, UK.

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Related in: MedlinePlus

Multicolor labeling tools in Drosophila. Transgenes following the excision-based Brainbow-1 strategy are highlighted in blue. Transgenes modeled on the inversion/excision-based Brainbow-2 strategy are shown in purple. Constructs are downstream of upstream activation sequences (UAS). (a) In dBrainbow, a stop cassette prevents marker expression prior to Cre activation. FPs are detected with three epitope-tags. Native fluorescence signals can be collected for EGFP and mKusabira Orange2 (mKO2); EBFP2 requires detection by immunolabeling (asterisk). (b) In Flybow, FPs are membrane-tethered using cd8 or myristoylation-palmitoylation (mp) sequences. Flybow B transgenes use mTurquoise (mTq) instead of V5-tagged mCerulean (mCer), which requires immunodetection (asterisk). Flybow-1.0, 1.1, 1.0B, and 1.1B transgenes show default expression of mCherry (mCher) or EGFP. Flybow-2.0 and 2.0B require FLP-mediated excision of a FRT-site flanked stop cassette. Recombination events between mFRT71 sites are triggered by mFLP5. (c-e) UAS-Brainbow, LOLLIbow, and UAS-Brainbow2.1R-2, are derived from the mouse Brainbow transgenes M and R. Recombination events are mediated by Cre. LOLLIbow relies on photo-activated split-Cre. p, palmitoylation signal. (f) In TIE-DYE, FLP mediates the excision of stop cassettes in three separate transgenes controlled by ubiquitin (ubi) or actin (act) enhancers. Gal4 leads to expression of mRFP1. lacZ requires detection with an antibody against βGal (asterisk). Seven color outcomes are possible for the combination of these markers, targeted by a nuclear localization signal (nls) or Histone-2A (H2A). (g) Raeppli transgenes are downstream of lexAop or UAS. Cre-mediated excision converts transgenes into exclusively Gal4 or LexA controlled constructs. FLP-mediated excision of a FRT-flanked stop cassette, enables ϕC31 transcription. Integrase expression is controlled by the full heat shock protein 70 (hsp70) promoter or UAS. This leads to recombination between the attB site and one of the four attP sites preceding each FP and to integrase self-excision. E2-Or, E2-Orange. FPs label cell nuclei in Raeppli-NLS, and cell membranes using a farnesylation (f) signal in Raeppli-CAAX. References for transgenes are provided in Table1.
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fig03: Multicolor labeling tools in Drosophila. Transgenes following the excision-based Brainbow-1 strategy are highlighted in blue. Transgenes modeled on the inversion/excision-based Brainbow-2 strategy are shown in purple. Constructs are downstream of upstream activation sequences (UAS). (a) In dBrainbow, a stop cassette prevents marker expression prior to Cre activation. FPs are detected with three epitope-tags. Native fluorescence signals can be collected for EGFP and mKusabira Orange2 (mKO2); EBFP2 requires detection by immunolabeling (asterisk). (b) In Flybow, FPs are membrane-tethered using cd8 or myristoylation-palmitoylation (mp) sequences. Flybow B transgenes use mTurquoise (mTq) instead of V5-tagged mCerulean (mCer), which requires immunodetection (asterisk). Flybow-1.0, 1.1, 1.0B, and 1.1B transgenes show default expression of mCherry (mCher) or EGFP. Flybow-2.0 and 2.0B require FLP-mediated excision of a FRT-site flanked stop cassette. Recombination events between mFRT71 sites are triggered by mFLP5. (c-e) UAS-Brainbow, LOLLIbow, and UAS-Brainbow2.1R-2, are derived from the mouse Brainbow transgenes M and R. Recombination events are mediated by Cre. LOLLIbow relies on photo-activated split-Cre. p, palmitoylation signal. (f) In TIE-DYE, FLP mediates the excision of stop cassettes in three separate transgenes controlled by ubiquitin (ubi) or actin (act) enhancers. Gal4 leads to expression of mRFP1. lacZ requires detection with an antibody against βGal (asterisk). Seven color outcomes are possible for the combination of these markers, targeted by a nuclear localization signal (nls) or Histone-2A (H2A). (g) Raeppli transgenes are downstream of lexAop or UAS. Cre-mediated excision converts transgenes into exclusively Gal4 or LexA controlled constructs. FLP-mediated excision of a FRT-flanked stop cassette, enables ϕC31 transcription. Integrase expression is controlled by the full heat shock protein 70 (hsp70) promoter or UAS. This leads to recombination between the attB site and one of the four attP sites preceding each FP and to integrase self-excision. E2-Or, E2-Orange. FPs label cell nuclei in Raeppli-NLS, and cell membranes using a farnesylation (f) signal in Raeppli-CAAX. References for transgenes are provided in Table1.

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


Versatile genetic paintbrushes: Brainbow technologies.

Richier B, Salecker I - Wiley Interdiscip Rev Dev Biol (2014)

Multicolor labeling tools in Drosophila. Transgenes following the excision-based Brainbow-1 strategy are highlighted in blue. Transgenes modeled on the inversion/excision-based Brainbow-2 strategy are shown in purple. Constructs are downstream of upstream activation sequences (UAS). (a) In dBrainbow, a stop cassette prevents marker expression prior to Cre activation. FPs are detected with three epitope-tags. Native fluorescence signals can be collected for EGFP and mKusabira Orange2 (mKO2); EBFP2 requires detection by immunolabeling (asterisk). (b) In Flybow, FPs are membrane-tethered using cd8 or myristoylation-palmitoylation (mp) sequences. Flybow B transgenes use mTurquoise (mTq) instead of V5-tagged mCerulean (mCer), which requires immunodetection (asterisk). Flybow-1.0, 1.1, 1.0B, and 1.1B transgenes show default expression of mCherry (mCher) or EGFP. Flybow-2.0 and 2.0B require FLP-mediated excision of a FRT-site flanked stop cassette. Recombination events between mFRT71 sites are triggered by mFLP5. (c-e) UAS-Brainbow, LOLLIbow, and UAS-Brainbow2.1R-2, are derived from the mouse Brainbow transgenes M and R. Recombination events are mediated by Cre. LOLLIbow relies on photo-activated split-Cre. p, palmitoylation signal. (f) In TIE-DYE, FLP mediates the excision of stop cassettes in three separate transgenes controlled by ubiquitin (ubi) or actin (act) enhancers. Gal4 leads to expression of mRFP1. lacZ requires detection with an antibody against βGal (asterisk). Seven color outcomes are possible for the combination of these markers, targeted by a nuclear localization signal (nls) or Histone-2A (H2A). (g) Raeppli transgenes are downstream of lexAop or UAS. Cre-mediated excision converts transgenes into exclusively Gal4 or LexA controlled constructs. FLP-mediated excision of a FRT-flanked stop cassette, enables ϕC31 transcription. Integrase expression is controlled by the full heat shock protein 70 (hsp70) promoter or UAS. This leads to recombination between the attB site and one of the four attP sites preceding each FP and to integrase self-excision. E2-Or, E2-Orange. FPs label cell nuclei in Raeppli-NLS, and cell membranes using a farnesylation (f) signal in Raeppli-CAAX. References for transgenes are provided in Table1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4384809&req=5

fig03: Multicolor labeling tools in Drosophila. Transgenes following the excision-based Brainbow-1 strategy are highlighted in blue. Transgenes modeled on the inversion/excision-based Brainbow-2 strategy are shown in purple. Constructs are downstream of upstream activation sequences (UAS). (a) In dBrainbow, a stop cassette prevents marker expression prior to Cre activation. FPs are detected with three epitope-tags. Native fluorescence signals can be collected for EGFP and mKusabira Orange2 (mKO2); EBFP2 requires detection by immunolabeling (asterisk). (b) In Flybow, FPs are membrane-tethered using cd8 or myristoylation-palmitoylation (mp) sequences. Flybow B transgenes use mTurquoise (mTq) instead of V5-tagged mCerulean (mCer), which requires immunodetection (asterisk). Flybow-1.0, 1.1, 1.0B, and 1.1B transgenes show default expression of mCherry (mCher) or EGFP. Flybow-2.0 and 2.0B require FLP-mediated excision of a FRT-site flanked stop cassette. Recombination events between mFRT71 sites are triggered by mFLP5. (c-e) UAS-Brainbow, LOLLIbow, and UAS-Brainbow2.1R-2, are derived from the mouse Brainbow transgenes M and R. Recombination events are mediated by Cre. LOLLIbow relies on photo-activated split-Cre. p, palmitoylation signal. (f) In TIE-DYE, FLP mediates the excision of stop cassettes in three separate transgenes controlled by ubiquitin (ubi) or actin (act) enhancers. Gal4 leads to expression of mRFP1. lacZ requires detection with an antibody against βGal (asterisk). Seven color outcomes are possible for the combination of these markers, targeted by a nuclear localization signal (nls) or Histone-2A (H2A). (g) Raeppli transgenes are downstream of lexAop or UAS. Cre-mediated excision converts transgenes into exclusively Gal4 or LexA controlled constructs. FLP-mediated excision of a FRT-flanked stop cassette, enables ϕC31 transcription. Integrase expression is controlled by the full heat shock protein 70 (hsp70) promoter or UAS. This leads to recombination between the attB site and one of the four attP sites preceding each FP and to integrase self-excision. E2-Or, E2-Orange. FPs label cell nuclei in Raeppli-NLS, and cell membranes using a farnesylation (f) signal in Raeppli-CAAX. References for transgenes are provided in Table1.
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

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.

View Article: PubMed Central - PubMed

Affiliation: MRC National Institute for Medical Research, Division of Molecular Neurobiology, London, UK.

Show MeSH
Related in: MedlinePlus