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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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Principles of mouse Brainbow blueprints. (a) Brainbow-1 strategy transgenes (blue) build on the ability of Cre to mediate excisions between heterospecific lox pairs orientated in the same direction. In Brainbow-1.0 (L), dTomato (dTom) is expressed by default. Cre catalyzes recombination events between lox2272 or loxP pairs, resulting in the stochastic expression of mCerulean (mCer) or mEYFP, respectively. In Brainbow-1.1 (M), the default marker is mKusabira Orange (mKO). Cre mediates recombination between loxN, lox2272, and loxP pairs, allowing the expression of mCherry (mCher), mEYFP or mCerulean. A palmitoylation signal (p) targets these FPs to the membrane. pA, polyadenylation signals. (b) Brainbow-2 strategy transgenes (purple) use the ability of Cre to mediate inversions and excisions between loxP sites oriented in the opposite and the same direction, respectively. Brainbow-2.0 consists of one invertible cassette. tdimer2 is expressed by default. Cre triggers reversible inversions between loxP sites, inducing expression of palmitoylated ECFP. Brainbow-2.1 (R) consists of two invertible cassettes. Nuclear (nls) GFP is expressed by default. Cre-mediated inversions and excisions between loxP pairs allow expression of mEYFP, tdimer2 or palmitoylated mCerulean. All transgenes are under the control of the nervous system specific Thy1 enhancer. (c) Combinatorial expression of blue, green, and red FPs from three transgene copies increases the color palette from 3 to 10 hues. References for transgenes are provided in Table1.
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fig02: Principles of mouse Brainbow blueprints. (a) Brainbow-1 strategy transgenes (blue) build on the ability of Cre to mediate excisions between heterospecific lox pairs orientated in the same direction. In Brainbow-1.0 (L), dTomato (dTom) is expressed by default. Cre catalyzes recombination events between lox2272 or loxP pairs, resulting in the stochastic expression of mCerulean (mCer) or mEYFP, respectively. In Brainbow-1.1 (M), the default marker is mKusabira Orange (mKO). Cre mediates recombination between loxN, lox2272, and loxP pairs, allowing the expression of mCherry (mCher), mEYFP or mCerulean. A palmitoylation signal (p) targets these FPs to the membrane. pA, polyadenylation signals. (b) Brainbow-2 strategy transgenes (purple) use the ability of Cre to mediate inversions and excisions between loxP sites oriented in the opposite and the same direction, respectively. Brainbow-2.0 consists of one invertible cassette. tdimer2 is expressed by default. Cre triggers reversible inversions between loxP sites, inducing expression of palmitoylated ECFP. Brainbow-2.1 (R) consists of two invertible cassettes. Nuclear (nls) GFP is expressed by default. Cre-mediated inversions and excisions between loxP pairs allow expression of mEYFP, tdimer2 or palmitoylated mCerulean. All transgenes are under the control of the nervous system specific Thy1 enhancer. (c) Combinatorial expression of blue, green, and red FPs from three transgene copies increases the color palette from 3 to 10 hues. References for transgenes are provided in Table1.

Mentions: Exploiting the expanding FP color palette and site-specific recombination technologies, multicolor labeling was first achieved by the Brainbow system devised by Livet et al. for mice.2 This creative approach takes advantage of the Cre-lox system to stochastically drive the expression of one of three or four FPs from a single transgene in genetically defined cell populations. Brainbow transgenes follow two principles. The Brainbow-1 strategy relies on Cre-mediated excision of DNA fragments using heterospecific lox sites (Figure 2(a)). In Brainbow-1.0 and -1.1 transgenes, three lox pairs (loxN, lox2272, and loxP) are astutely positioned in the same orientation adjacent to three or four linearly arranged FP-encoding cDNAs. These are each followed by polyA termination sequences to prevent transcriptional read-through. The FP located closest to the promoter is expressed by default. Upon Cre activation, site-specific recombination between identical lox pairs causes the excision of one, two, or three FP sequences. Consequently, new FPs are randomly positioned closest to the promoter. This leads to the stable, mutually exclusive expression of one of three or four FPs per cell in a tissue. By contrast, the Brainbow-2 strategy makes use of inversion and excision events between a single type of recombination site, loxP (Figure 2(b)). The coding sequences of two FPs are arranged in opposite orientations in an invertible cassette flanked by inward-facing loxP sites. Brainbow-2.0 contains one such cassette, and Cre-mediated inversion results in the differential expression of two markers. Brainbow-2.1 transgenes consist of two adjacent cassettes. Recombination of loxP pairs in opposite or identical orientation leads to inversion and excision of cassettes, respectively. This results in four color-outcomes. Because inversions are reversible, transient Cre expression is required. Brainbow transgenes are controlled by the Thy-1 enhancer to activate expression in neurons or glia, while recombination events are mediated by ubiquitous or tissue-specific Cre transgenes.


Versatile genetic paintbrushes: Brainbow technologies.

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

Principles of mouse Brainbow blueprints. (a) Brainbow-1 strategy transgenes (blue) build on the ability of Cre to mediate excisions between heterospecific lox pairs orientated in the same direction. In Brainbow-1.0 (L), dTomato (dTom) is expressed by default. Cre catalyzes recombination events between lox2272 or loxP pairs, resulting in the stochastic expression of mCerulean (mCer) or mEYFP, respectively. In Brainbow-1.1 (M), the default marker is mKusabira Orange (mKO). Cre mediates recombination between loxN, lox2272, and loxP pairs, allowing the expression of mCherry (mCher), mEYFP or mCerulean. A palmitoylation signal (p) targets these FPs to the membrane. pA, polyadenylation signals. (b) Brainbow-2 strategy transgenes (purple) use the ability of Cre to mediate inversions and excisions between loxP sites oriented in the opposite and the same direction, respectively. Brainbow-2.0 consists of one invertible cassette. tdimer2 is expressed by default. Cre triggers reversible inversions between loxP sites, inducing expression of palmitoylated ECFP. Brainbow-2.1 (R) consists of two invertible cassettes. Nuclear (nls) GFP is expressed by default. Cre-mediated inversions and excisions between loxP pairs allow expression of mEYFP, tdimer2 or palmitoylated mCerulean. All transgenes are under the control of the nervous system specific Thy1 enhancer. (c) Combinatorial expression of blue, green, and red FPs from three transgene copies increases the color palette from 3 to 10 hues. References for transgenes are provided in Table1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig02: Principles of mouse Brainbow blueprints. (a) Brainbow-1 strategy transgenes (blue) build on the ability of Cre to mediate excisions between heterospecific lox pairs orientated in the same direction. In Brainbow-1.0 (L), dTomato (dTom) is expressed by default. Cre catalyzes recombination events between lox2272 or loxP pairs, resulting in the stochastic expression of mCerulean (mCer) or mEYFP, respectively. In Brainbow-1.1 (M), the default marker is mKusabira Orange (mKO). Cre mediates recombination between loxN, lox2272, and loxP pairs, allowing the expression of mCherry (mCher), mEYFP or mCerulean. A palmitoylation signal (p) targets these FPs to the membrane. pA, polyadenylation signals. (b) Brainbow-2 strategy transgenes (purple) use the ability of Cre to mediate inversions and excisions between loxP sites oriented in the opposite and the same direction, respectively. Brainbow-2.0 consists of one invertible cassette. tdimer2 is expressed by default. Cre triggers reversible inversions between loxP sites, inducing expression of palmitoylated ECFP. Brainbow-2.1 (R) consists of two invertible cassettes. Nuclear (nls) GFP is expressed by default. Cre-mediated inversions and excisions between loxP pairs allow expression of mEYFP, tdimer2 or palmitoylated mCerulean. All transgenes are under the control of the nervous system specific Thy1 enhancer. (c) Combinatorial expression of blue, green, and red FPs from three transgene copies increases the color palette from 3 to 10 hues. References for transgenes are provided in Table1.
Mentions: Exploiting the expanding FP color palette and site-specific recombination technologies, multicolor labeling was first achieved by the Brainbow system devised by Livet et al. for mice.2 This creative approach takes advantage of the Cre-lox system to stochastically drive the expression of one of three or four FPs from a single transgene in genetically defined cell populations. Brainbow transgenes follow two principles. The Brainbow-1 strategy relies on Cre-mediated excision of DNA fragments using heterospecific lox sites (Figure 2(a)). In Brainbow-1.0 and -1.1 transgenes, three lox pairs (loxN, lox2272, and loxP) are astutely positioned in the same orientation adjacent to three or four linearly arranged FP-encoding cDNAs. These are each followed by polyA termination sequences to prevent transcriptional read-through. The FP located closest to the promoter is expressed by default. Upon Cre activation, site-specific recombination between identical lox pairs causes the excision of one, two, or three FP sequences. Consequently, new FPs are randomly positioned closest to the promoter. This leads to the stable, mutually exclusive expression of one of three or four FPs per cell in a tissue. By contrast, the Brainbow-2 strategy makes use of inversion and excision events between a single type of recombination site, loxP (Figure 2(b)). The coding sequences of two FPs are arranged in opposite orientations in an invertible cassette flanked by inward-facing loxP sites. Brainbow-2.0 contains one such cassette, and Cre-mediated inversion results in the differential expression of two markers. Brainbow-2.1 transgenes consist of two adjacent cassettes. Recombination of loxP pairs in opposite or identical orientation leads to inversion and excision of cassettes, respectively. This results in four color-outcomes. Because inversions are reversible, transient Cre expression is required. Brainbow transgenes are controlled by the Thy-1 enhancer to activate expression in neurons or glia, while recombination events are mediated by ubiquitous or tissue-specific Cre transgenes.

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