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Improved tools for the Brainbow toolbox.

Cai D, Cohen KB, Luo T, Lichtman JW, Sanes JR - Nat. Methods (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 1] Center for Brain Science, Harvard University, Cambridge, Massachusetts, USA. [2] Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.

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.

No MeSH data available.


Related in: MedlinePlus

Brainbow AAV(a) AAV Brainbow constructs and recombination scheme. Farnesylated TagBFP and EYFP or mCherry and mTFP were placed in reverse orientation between mutant Lox sites. EF1α, regulatory elements from elongation factor 1α gene; W, WPRE; triangles, Lox mutants and their recombination products; dark and light sectors indicate wild-type and mutant portions of Lox sites, respectively; 1–6, recombination events.(b) Outcomes of recombination events numbered in a. Open arrows, direction of intervening cDNA; X, fully mutant (light green) lox site cannot serve as substrate for Cre.(c) Eight color outcomes resulting from pairs of Brainbow AAVs following recombination as shown in a. This is a minimum value, because it does not account for differences in relative intensity of the four XFPs(d,e) Test of color balance. The mTFPf-mCherryf AAV vector was injected at low titer into the cortex of a Thy1-Cre mouse and neurons of each color were counted. Similar fractions of neurons expressed mTFPf (58%) and mCherryf (42%; n=1523 neurons in 4 sections of three mice; red shows standard deviation.)(f–h) AAV injected into cortex of PV-cre mice predominantly labels interneurons. g and h, high magnification views of boxed regions in f.(i) AAV injected into retina of mouse expressing Cre in retinal ganglion cell subset shows labeling of ganglion cell somata, dendrites (arrow) and axons (arrowhead).(j) AAV injected into cerebellum of PV-Cre mouse labels Purkinje cells (fan shaped dendrites, white arrows) and interneurons (cell bodies and axons, yellow arrows).In f–j antibody amplified mTFP and EYFP were in green; TagBFP was in blue and mCherry was in red.Bars are 20µm in f, 10µm in g and h, 50µm in i and j
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Figure 5: Brainbow AAV(a) AAV Brainbow constructs and recombination scheme. Farnesylated TagBFP and EYFP or mCherry and mTFP were placed in reverse orientation between mutant Lox sites. EF1α, regulatory elements from elongation factor 1α gene; W, WPRE; triangles, Lox mutants and their recombination products; dark and light sectors indicate wild-type and mutant portions of Lox sites, respectively; 1–6, recombination events.(b) Outcomes of recombination events numbered in a. Open arrows, direction of intervening cDNA; X, fully mutant (light green) lox site cannot serve as substrate for Cre.(c) Eight color outcomes resulting from pairs of Brainbow AAVs following recombination as shown in a. This is a minimum value, because it does not account for differences in relative intensity of the four XFPs(d,e) Test of color balance. The mTFPf-mCherryf AAV vector was injected at low titer into the cortex of a Thy1-Cre mouse and neurons of each color were counted. Similar fractions of neurons expressed mTFPf (58%) and mCherryf (42%; n=1523 neurons in 4 sections of three mice; red shows standard deviation.)(f–h) AAV injected into cortex of PV-cre mice predominantly labels interneurons. g and h, high magnification views of boxed regions in f.(i) AAV injected into retina of mouse expressing Cre in retinal ganglion cell subset shows labeling of ganglion cell somata, dendrites (arrow) and axons (arrowhead).(j) AAV injected into cerebellum of PV-Cre mouse labels Purkinje cells (fan shaped dendrites, white arrows) and interneurons (cell bodies and axons, yellow arrows).In f–j antibody amplified mTFP and EYFP were in green; TagBFP was in blue and mCherry was in red.Bars are 20µm in f, 10µm in g and h, 50µm in i and j

Mentions: In parallel to generation of Brainbow transgenic lines, we generated adeno-associated viral (AAV) vectors to provide spatial and temporal control over expression and to make the method applicable to other species. Because the Brainbow 3.1 cassette described above is >6kB but the capacity of AAV vectors is <5kB, we reengineered the cassette. Based on results from initial tests illustrated in Supplementary Fig. 8, we devised a scheme in which lox sites with left or right element mutations44 were used for unidirectional Cre-dependent inversion (Fig. 5a,b). Farnesylated XFPs were positioned in reverse orientation to prevent Cre-independent expression, and WPRE elements were added to increase expression. In this design, recombination can lead to three outcomes from two XFPs: XFP1, XFP2 or neither. We generated two AAVs with 2 XFPs each, such that co-infection would lead to a minimum of 8 hues (3 × 3 −1; Fig. 5c). Because AAV can infect cells at high multiplicity, the number of possible colors is >>8. An additional feature is that excision of the non-expressed XFP in a second step (#3 and #4 in Fig. 5a) enhances and equalizes expression of the remaining XFP (Fig. 5d,e).


Improved tools for the Brainbow toolbox.

Cai D, Cohen KB, Luo T, Lichtman JW, Sanes JR - Nat. Methods (2013)

Brainbow AAV(a) AAV Brainbow constructs and recombination scheme. Farnesylated TagBFP and EYFP or mCherry and mTFP were placed in reverse orientation between mutant Lox sites. EF1α, regulatory elements from elongation factor 1α gene; W, WPRE; triangles, Lox mutants and their recombination products; dark and light sectors indicate wild-type and mutant portions of Lox sites, respectively; 1–6, recombination events.(b) Outcomes of recombination events numbered in a. Open arrows, direction of intervening cDNA; X, fully mutant (light green) lox site cannot serve as substrate for Cre.(c) Eight color outcomes resulting from pairs of Brainbow AAVs following recombination as shown in a. This is a minimum value, because it does not account for differences in relative intensity of the four XFPs(d,e) Test of color balance. The mTFPf-mCherryf AAV vector was injected at low titer into the cortex of a Thy1-Cre mouse and neurons of each color were counted. Similar fractions of neurons expressed mTFPf (58%) and mCherryf (42%; n=1523 neurons in 4 sections of three mice; red shows standard deviation.)(f–h) AAV injected into cortex of PV-cre mice predominantly labels interneurons. g and h, high magnification views of boxed regions in f.(i) AAV injected into retina of mouse expressing Cre in retinal ganglion cell subset shows labeling of ganglion cell somata, dendrites (arrow) and axons (arrowhead).(j) AAV injected into cerebellum of PV-Cre mouse labels Purkinje cells (fan shaped dendrites, white arrows) and interneurons (cell bodies and axons, yellow arrows).In f–j antibody amplified mTFP and EYFP were in green; TagBFP was in blue and mCherry was in red.Bars are 20µm in f, 10µm in g and h, 50µm in i and j
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Related In: Results  -  Collection

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Figure 5: Brainbow AAV(a) AAV Brainbow constructs and recombination scheme. Farnesylated TagBFP and EYFP or mCherry and mTFP were placed in reverse orientation between mutant Lox sites. EF1α, regulatory elements from elongation factor 1α gene; W, WPRE; triangles, Lox mutants and their recombination products; dark and light sectors indicate wild-type and mutant portions of Lox sites, respectively; 1–6, recombination events.(b) Outcomes of recombination events numbered in a. Open arrows, direction of intervening cDNA; X, fully mutant (light green) lox site cannot serve as substrate for Cre.(c) Eight color outcomes resulting from pairs of Brainbow AAVs following recombination as shown in a. This is a minimum value, because it does not account for differences in relative intensity of the four XFPs(d,e) Test of color balance. The mTFPf-mCherryf AAV vector was injected at low titer into the cortex of a Thy1-Cre mouse and neurons of each color were counted. Similar fractions of neurons expressed mTFPf (58%) and mCherryf (42%; n=1523 neurons in 4 sections of three mice; red shows standard deviation.)(f–h) AAV injected into cortex of PV-cre mice predominantly labels interneurons. g and h, high magnification views of boxed regions in f.(i) AAV injected into retina of mouse expressing Cre in retinal ganglion cell subset shows labeling of ganglion cell somata, dendrites (arrow) and axons (arrowhead).(j) AAV injected into cerebellum of PV-Cre mouse labels Purkinje cells (fan shaped dendrites, white arrows) and interneurons (cell bodies and axons, yellow arrows).In f–j antibody amplified mTFP and EYFP were in green; TagBFP was in blue and mCherry was in red.Bars are 20µm in f, 10µm in g and h, 50µm in i and j
Mentions: In parallel to generation of Brainbow transgenic lines, we generated adeno-associated viral (AAV) vectors to provide spatial and temporal control over expression and to make the method applicable to other species. Because the Brainbow 3.1 cassette described above is >6kB but the capacity of AAV vectors is <5kB, we reengineered the cassette. Based on results from initial tests illustrated in Supplementary Fig. 8, we devised a scheme in which lox sites with left or right element mutations44 were used for unidirectional Cre-dependent inversion (Fig. 5a,b). Farnesylated XFPs were positioned in reverse orientation to prevent Cre-independent expression, and WPRE elements were added to increase expression. In this design, recombination can lead to three outcomes from two XFPs: XFP1, XFP2 or neither. We generated two AAVs with 2 XFPs each, such that co-infection would lead to a minimum of 8 hues (3 × 3 −1; Fig. 5c). Because AAV can infect cells at high multiplicity, the number of possible colors is >>8. An additional feature is that excision of the non-expressed XFP in a second step (#3 and #4 in Fig. 5a) enhances and equalizes expression of the remaining XFP (Fig. 5d,e).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 1] Center for Brain Science, Harvard University, Cambridge, Massachusetts, USA. [2] Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.

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.

No MeSH data available.


Related in: MedlinePlus