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Versatile genetic paintbrushes: Brainbow technologies.

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

Bottom Line: Multicolor cell labeling techniques have been successfully applied in studies analyzing the cellular components of neural circuits and other tissues, and the compositions and interactions of lineages.While being continuously refined, Brainbow technologies have thus found a firm place in the genetic toolboxes of developmental and neurobiologists.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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Multicolor labeling tools for use in zebrafish and mouse, as well as for electroporation in mouse and chicken. Constructs following the Brainbow-1 strategy are indicated in blue and constructs based on the Brainbow-2 strategy in purple. (a) In zebrafish, the mouse Brainbow-1.0L cassette has been placed downstream of four regulatory elements: the cytomegalovirus enhancer (CMV), the βactin2 (βact2) enhancer, upstream activation sequences (UAS), or the ubiquitin (ubi) enhancer. UAS-Zebrabow-B uses non-repetitive (nr) tandem UAS sites. V, variegated; B, broad; S, single; M, multiple. (b) In Confetti, a stop cassette precedes the two invertible cassettes of the original mouse Brainbow-2.1 (R) transgene. Expression is controlled by CAG, the chicken β-actin promoter with cytomegalovirus (CAG) enhancer. Grey lines only indicate a subset of possible recombination events. Thy1-controlled Brainbow-3.0, 3.1, 3.2 trangenes use farnesylated (f) FPs – mOrange2 (mO2), EGFP, and mKate2. In Brainbow-3.1 and 3.2, a stop cassette prevents default FP expression. In Brainbow-3.2, a woodchuck hepatitis virus posttranscriptional regulatory element (W) has been placed downstream of each FP. In Autobow, Thy1 controls expression of lox-flanked Cre to trigger recombination events and self-excision. In Flpbow-3.0 and 3.1 transgenes, FLP mediates recombination of spacer variant pairs FRT3, FRT5T2, and FRT545. Flpbow-3.1 uses a stop cassette and FP fused with a SUMOstar tag (S) for immunodetection. mCer, mCerulean; PhiYFP, Phialidium YFP; tdTom, tandem dimer Tomato. (c) MAGIC and CLoNe plasmids are transposon-based vectors suitable for electroporation experiments in mouse and chicken. Tol2 or PiggyBac (PB) transposases promote the genomic integration of vectors. Ubiquitous or tissue/cell-type specific Cre is provided by co-injected vectors (chick and mouse) or by expression from genomic insertions (mouse). FP expression is controlled by the CAG regulatory element. In MAGIC markers, four different FPs are expressed from single vectors. FP are localized in the cytoplasm (Cytbow), in nuclei using Histone-2B (H2B) fusions (Nucbow), in the membrane using a palmitoylation (p) signal (Palmbow) or in mitochondria (mit) using a targeting signal from human COX8 (Mitbow). H2B-EBFP2 is expressed in unrecombined cells. In the CLoNe approach, four FPs [EGFP, mT-Sapphire (mT-Sap), mEYFP, and mCherry] are expressed from twelve separate labeling vectors. FPs are either cytoplasmic (cy), or nuclear (nc), and membrane-bound (mb) using H2B or palmitoylation tags, respectively. A stop cassette prevents default expression of markers in the absence of Cre. Stable multicolor labeling is achieved by different random combinations of vector insertions and expression in individual cells. Asterisk indicates that mT-Sapphire was assigned the color blue, although the maximum emission is in the green/yellow range. References for transgenes are provided in Table1.
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fig04: Multicolor labeling tools for use in zebrafish and mouse, as well as for electroporation in mouse and chicken. Constructs following the Brainbow-1 strategy are indicated in blue and constructs based on the Brainbow-2 strategy in purple. (a) In zebrafish, the mouse Brainbow-1.0L cassette has been placed downstream of four regulatory elements: the cytomegalovirus enhancer (CMV), the βactin2 (βact2) enhancer, upstream activation sequences (UAS), or the ubiquitin (ubi) enhancer. UAS-Zebrabow-B uses non-repetitive (nr) tandem UAS sites. V, variegated; B, broad; S, single; M, multiple. (b) In Confetti, a stop cassette precedes the two invertible cassettes of the original mouse Brainbow-2.1 (R) transgene. Expression is controlled by CAG, the chicken β-actin promoter with cytomegalovirus (CAG) enhancer. Grey lines only indicate a subset of possible recombination events. Thy1-controlled Brainbow-3.0, 3.1, 3.2 trangenes use farnesylated (f) FPs – mOrange2 (mO2), EGFP, and mKate2. In Brainbow-3.1 and 3.2, a stop cassette prevents default FP expression. In Brainbow-3.2, a woodchuck hepatitis virus posttranscriptional regulatory element (W) has been placed downstream of each FP. In Autobow, Thy1 controls expression of lox-flanked Cre to trigger recombination events and self-excision. In Flpbow-3.0 and 3.1 transgenes, FLP mediates recombination of spacer variant pairs FRT3, FRT5T2, and FRT545. Flpbow-3.1 uses a stop cassette and FP fused with a SUMOstar tag (S) for immunodetection. mCer, mCerulean; PhiYFP, Phialidium YFP; tdTom, tandem dimer Tomato. (c) MAGIC and CLoNe plasmids are transposon-based vectors suitable for electroporation experiments in mouse and chicken. Tol2 or PiggyBac (PB) transposases promote the genomic integration of vectors. Ubiquitous or tissue/cell-type specific Cre is provided by co-injected vectors (chick and mouse) or by expression from genomic insertions (mouse). FP expression is controlled by the CAG regulatory element. In MAGIC markers, four different FPs are expressed from single vectors. FP are localized in the cytoplasm (Cytbow), in nuclei using Histone-2B (H2B) fusions (Nucbow), in the membrane using a palmitoylation (p) signal (Palmbow) or in mitochondria (mit) using a targeting signal from human COX8 (Mitbow). H2B-EBFP2 is expressed in unrecombined cells. In the CLoNe approach, four FPs [EGFP, mT-Sapphire (mT-Sap), mEYFP, and mCherry] are expressed from twelve separate labeling vectors. FPs are either cytoplasmic (cy), or nuclear (nc), and membrane-bound (mb) using H2B or palmitoylation tags, respectively. A stop cassette prevents default expression of markers in the absence of Cre. Stable multicolor labeling is achieved by different random combinations of vector insertions and expression in individual cells. Asterisk indicates that mT-Sapphire was assigned the color blue, although the maximum emission is in the green/yellow range. References for transgenes are provided in Table1.

Mentions: Multicolor labeling approaches developed for zebrafish utilize the mouse Brainbow-1.0 (L) cassette (Figure 4(a)). In zebrafish Brainbow, this cassette is positioned downstream of the cytomegalovirus (CMV) promoter in a commonly used expression vector, that allows transient ubiquitous expression after injection.11βactin2-Brainbow goes one step further by using an actin enhancer in a stable transgenic line.12 In UAS-Brainbow-1.0L,13 and Zebrabow,14 tissue-specific transgene expression is controlled either by 14 UAS repeats (UAS-Brainbow1.0L with at least 3 insertions and UAS-Zebrabow-V with 2 insertions) or four non-repetitive UAS sequences (UAS-Zebrabow-B with 9–31 copies). In the presence of Gal4, the former results in variegated mosaic expression because repeat sequences are prone to CpG methylation and randomly silenced,95 thus rendering this strategy highly suitable for sparse labeling. By contrast, tandem UAS sites with four unique sequences are less susceptible to methylation, thus enabling broad cell labeling. Additional Zebrabow transgenes are controlled by the ubiquitin enhancer and either contain a single (ubi-Zebrabow-S) or multiple (16–32) insertions (ubi-Zebrabow-M).14 While transgenic lines with a high number of insertions in principle produce many hues, data analysis may be limited by the capacity of image processing software in achieving the necessary resolution of colors. Site-specific recombination events are mediated by Cre, which can be delivered by microinjection of a purified protein. Alternatively, zebrafish transgenics can be crossed with heat shock-inducible Cre or tamoxifen-inducible CreER lines that either are widely expressed or controlled by tissue-specific enhancers.14


Versatile genetic paintbrushes: Brainbow technologies.

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

Multicolor labeling tools for use in zebrafish and mouse, as well as for electroporation in mouse and chicken. Constructs following the Brainbow-1 strategy are indicated in blue and constructs based on the Brainbow-2 strategy in purple. (a) In zebrafish, the mouse Brainbow-1.0L cassette has been placed downstream of four regulatory elements: the cytomegalovirus enhancer (CMV), the βactin2 (βact2) enhancer, upstream activation sequences (UAS), or the ubiquitin (ubi) enhancer. UAS-Zebrabow-B uses non-repetitive (nr) tandem UAS sites. V, variegated; B, broad; S, single; M, multiple. (b) In Confetti, a stop cassette precedes the two invertible cassettes of the original mouse Brainbow-2.1 (R) transgene. Expression is controlled by CAG, the chicken β-actin promoter with cytomegalovirus (CAG) enhancer. Grey lines only indicate a subset of possible recombination events. Thy1-controlled Brainbow-3.0, 3.1, 3.2 trangenes use farnesylated (f) FPs – mOrange2 (mO2), EGFP, and mKate2. In Brainbow-3.1 and 3.2, a stop cassette prevents default FP expression. In Brainbow-3.2, a woodchuck hepatitis virus posttranscriptional regulatory element (W) has been placed downstream of each FP. In Autobow, Thy1 controls expression of lox-flanked Cre to trigger recombination events and self-excision. In Flpbow-3.0 and 3.1 transgenes, FLP mediates recombination of spacer variant pairs FRT3, FRT5T2, and FRT545. Flpbow-3.1 uses a stop cassette and FP fused with a SUMOstar tag (S) for immunodetection. mCer, mCerulean; PhiYFP, Phialidium YFP; tdTom, tandem dimer Tomato. (c) MAGIC and CLoNe plasmids are transposon-based vectors suitable for electroporation experiments in mouse and chicken. Tol2 or PiggyBac (PB) transposases promote the genomic integration of vectors. Ubiquitous or tissue/cell-type specific Cre is provided by co-injected vectors (chick and mouse) or by expression from genomic insertions (mouse). FP expression is controlled by the CAG regulatory element. In MAGIC markers, four different FPs are expressed from single vectors. FP are localized in the cytoplasm (Cytbow), in nuclei using Histone-2B (H2B) fusions (Nucbow), in the membrane using a palmitoylation (p) signal (Palmbow) or in mitochondria (mit) using a targeting signal from human COX8 (Mitbow). H2B-EBFP2 is expressed in unrecombined cells. In the CLoNe approach, four FPs [EGFP, mT-Sapphire (mT-Sap), mEYFP, and mCherry] are expressed from twelve separate labeling vectors. FPs are either cytoplasmic (cy), or nuclear (nc), and membrane-bound (mb) using H2B or palmitoylation tags, respectively. A stop cassette prevents default expression of markers in the absence of Cre. Stable multicolor labeling is achieved by different random combinations of vector insertions and expression in individual cells. Asterisk indicates that mT-Sapphire was assigned the color blue, although the maximum emission is in the green/yellow range. 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

fig04: Multicolor labeling tools for use in zebrafish and mouse, as well as for electroporation in mouse and chicken. Constructs following the Brainbow-1 strategy are indicated in blue and constructs based on the Brainbow-2 strategy in purple. (a) In zebrafish, the mouse Brainbow-1.0L cassette has been placed downstream of four regulatory elements: the cytomegalovirus enhancer (CMV), the βactin2 (βact2) enhancer, upstream activation sequences (UAS), or the ubiquitin (ubi) enhancer. UAS-Zebrabow-B uses non-repetitive (nr) tandem UAS sites. V, variegated; B, broad; S, single; M, multiple. (b) In Confetti, a stop cassette precedes the two invertible cassettes of the original mouse Brainbow-2.1 (R) transgene. Expression is controlled by CAG, the chicken β-actin promoter with cytomegalovirus (CAG) enhancer. Grey lines only indicate a subset of possible recombination events. Thy1-controlled Brainbow-3.0, 3.1, 3.2 trangenes use farnesylated (f) FPs – mOrange2 (mO2), EGFP, and mKate2. In Brainbow-3.1 and 3.2, a stop cassette prevents default FP expression. In Brainbow-3.2, a woodchuck hepatitis virus posttranscriptional regulatory element (W) has been placed downstream of each FP. In Autobow, Thy1 controls expression of lox-flanked Cre to trigger recombination events and self-excision. In Flpbow-3.0 and 3.1 transgenes, FLP mediates recombination of spacer variant pairs FRT3, FRT5T2, and FRT545. Flpbow-3.1 uses a stop cassette and FP fused with a SUMOstar tag (S) for immunodetection. mCer, mCerulean; PhiYFP, Phialidium YFP; tdTom, tandem dimer Tomato. (c) MAGIC and CLoNe plasmids are transposon-based vectors suitable for electroporation experiments in mouse and chicken. Tol2 or PiggyBac (PB) transposases promote the genomic integration of vectors. Ubiquitous or tissue/cell-type specific Cre is provided by co-injected vectors (chick and mouse) or by expression from genomic insertions (mouse). FP expression is controlled by the CAG regulatory element. In MAGIC markers, four different FPs are expressed from single vectors. FP are localized in the cytoplasm (Cytbow), in nuclei using Histone-2B (H2B) fusions (Nucbow), in the membrane using a palmitoylation (p) signal (Palmbow) or in mitochondria (mit) using a targeting signal from human COX8 (Mitbow). H2B-EBFP2 is expressed in unrecombined cells. In the CLoNe approach, four FPs [EGFP, mT-Sapphire (mT-Sap), mEYFP, and mCherry] are expressed from twelve separate labeling vectors. FPs are either cytoplasmic (cy), or nuclear (nc), and membrane-bound (mb) using H2B or palmitoylation tags, respectively. A stop cassette prevents default expression of markers in the absence of Cre. Stable multicolor labeling is achieved by different random combinations of vector insertions and expression in individual cells. Asterisk indicates that mT-Sapphire was assigned the color blue, although the maximum emission is in the green/yellow range. References for transgenes are provided in Table1.
Mentions: Multicolor labeling approaches developed for zebrafish utilize the mouse Brainbow-1.0 (L) cassette (Figure 4(a)). In zebrafish Brainbow, this cassette is positioned downstream of the cytomegalovirus (CMV) promoter in a commonly used expression vector, that allows transient ubiquitous expression after injection.11βactin2-Brainbow goes one step further by using an actin enhancer in a stable transgenic line.12 In UAS-Brainbow-1.0L,13 and Zebrabow,14 tissue-specific transgene expression is controlled either by 14 UAS repeats (UAS-Brainbow1.0L with at least 3 insertions and UAS-Zebrabow-V with 2 insertions) or four non-repetitive UAS sequences (UAS-Zebrabow-B with 9–31 copies). In the presence of Gal4, the former results in variegated mosaic expression because repeat sequences are prone to CpG methylation and randomly silenced,95 thus rendering this strategy highly suitable for sparse labeling. By contrast, tandem UAS sites with four unique sequences are less susceptible to methylation, thus enabling broad cell labeling. Additional Zebrabow transgenes are controlled by the ubiquitin enhancer and either contain a single (ubi-Zebrabow-S) or multiple (16–32) insertions (ubi-Zebrabow-M).14 While transgenic lines with a high number of insertions in principle produce many hues, data analysis may be limited by the capacity of image processing software in achieving the necessary resolution of colors. Site-specific recombination events are mediated by Cre, which can be delivered by microinjection of a purified protein. Alternatively, zebrafish transgenics can be crossed with heat shock-inducible Cre or tamoxifen-inducible CreER lines that either are widely expressed or controlled by tissue-specific enhancers.14

Bottom Line: Multicolor cell labeling techniques have been successfully applied in studies analyzing the cellular components of neural circuits and other tissues, and the compositions and interactions of lineages.While being continuously refined, Brainbow technologies have thus found a firm place in the genetic toolboxes of developmental and neurobiologists.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