B6.Cg-Tg(Thy1-Brainbow2.1)RLich/J

Stock number: 007921
Research areas: Neurobiology Research, Research Tools

Description

These Brainbow 2.1 (founder line R) mice allow labeling of individual neurons in peripheral and central neurons with nuclear localized, membrane-targeted, or cytoplasmic fluorescent proteins in cre recombined cells, and may also be useful in conjunction with other Brainbow strains (Stock No. 007901, Stock No. 007910, Stock No. 007911) for neurobiological studies.

Detailed description

These Thy1-Brainbow 2.1 (line R) transgenic mice are viable and fertile. The mice possess two invertible DNA segments (four fluorescent protein sequences in total) uniquely positioned in tandem and flanked with LoxP sites to generate a larger number of recombination outcomes; allowing stochastic expression of multiple fluorescent proteins from a single transgene. Although the fluorescent protein immediately adjacent to the promoter, hrGFPII (with nuclear localization signal), was designed to be expressed prior to Cre-mediated recombination, basal hrGFPII expression may not be observed in mouse tissues. When bred to Cre recombinase expressing mice, however, the resulting offspring can have one of three different inversions for each transgene in each cell of the cre expressing tissue(s). In addition, two excision events may reduce the construct to one of two single invertible DNA segments which can continue to invert as long as cre is present. These different recombination possibilities can be used to express four genes (hrGFPII, mYFP, tdimer2(12) (RFP), or mCerulean (CFP)) to the peripheral and central neurons. A nuclear localization signal targets the hrGFPII to the nucleus, a palmitoylation sequence tethers the mCerulean (CFP) to the membrane (allowing clear labeling of axonal processes), and cytoplasmic tdimer2(12) (RFP) and mYFP better labeled neuronal cell bodies and dendrites. Integration of tandem transgene copies yields combinatorial fluorescent protein expression in each cell, and thus many possible cell colors, providing a way to distinguish adjacent neurons and visualize other cellular interactions. Of note, the single FRT site inserted at the 5' end of the transgene allows tandem transgene copy number reduction through Flp-mediated recombination if desired. These Brainbow 2.1 (founder line R) mice allow labeling of individual neurons in peripheral and central neurons with nuclear localized, membrane-targeted, or cytoplasmic fluorescent proteins in cre recombined cells, and may also be useful in conjunction with other Brainbow strains (Stock No. 007901, Stock No. 007910, Stock No. 007911) for neurobiological studies.

View Brainbow images for Stock No's 007901, 007910, 007911, and 007921.

Development

The Thy1-Brainbow 2.1 transgene was designed with the mouse Thy1 regulatory elements surrounding four fluorescent proteins (green (GFP), enhanced yellow (EYFP), red (RFP), and cyan (CFP)) sequences uniquely positioned in a tandem fashion and delimited by LoxP sites in opposite orientation. Specifically, this Brainbow 2.1 coding region is composed of two adjacent head to tail tandem dimers, the first contains a loxP site, humanized Renilla GFP (hrGFPII (Stratagene); with nuclear localization signal), and polyA sequence in forward orientation, and a LoxP site, monomeric EYFP (mYFP^A206K), and polyA sequence in reverse orientation. The second head to tail dimer contains a LoxP site, tdimer2(12) RFP, and polyA sequence in forward orientation, and a LoxP site, mCerulean CFP (with membrane tethering palmitoylation sequence), and polyA sequence in reverse orientation. A single FRT site is located at the 3' end of the coding region. This transgene was microinjected into C57BL/6JxCBA hybrid oocytes. Founder mice (line R) were bred with C57BL/6. The resulting mutant mice were backcrossed to C57BL/6 for at least 5 generations prior to arrival at The Jackson Laboratory. Upon arrival, mice were bred to C57BL/6J (Stock No. 000664) for at least one generation to establish the colony.

The hrGFPII variant of GFP (from Stratagene vector phrGFPII-C) has amino acid substitutions designed to improve spectral properties and performance in mammalian systems. The monomeric EYFP (mYFP^A206K) has an amino acid substitution replacing a hydrophobic region with a positively charged residue designed to prevent dimerization. The tdimer2(12) RFP is a non-oligomerizing DsRed variant with a 12 residue linker fusing two copies of the protein (tandem dimer). The monomeric Cerulean (mCerulean) is a variant of ECFP (ECFP^{S72A/Y145A/H148D/A206K}) with amino acid substitutions designed to improve spectral properties and prevent dimerization.

Allele

Symbol: Tg(Thy1-Brainbow2.1)RLich
Name: transgene insertion R, Jeff W Lichtman
MGI accession ID: MGI:3761284
Generation method: Transgenic
Attributes: Reporter
Marker symbol: Tg(Thy1-Brainbow2.1)RLich
Marker name: transgene insertion R, Jeff W Lichtman
Chromosome: UN
Strain of origin: C57BL/6J x CBA
Expressed genes: GFP,Green Fluorescent Protein,; YFP,Yellow Fluorescent Protein,jellyfish; RFP,Red Fluorescent Protein,coral; CFP,Cyan Fluorescent Protein,jellyfish
Site of expression: When bred to Cre recombinase expressing mice, there is one of three different inversions for each transgene in each cell of the cre expressing tissue(s). In addition, two excision events may reduce the construct to one of two single invertible DNA segments which can continue to invert as long as cre is present. These different recombination possibilities can be used to express four genes (hrGFPII, mYFP, tdimer2(12) (RFP), or mCerulean (CFP)). Integration of multiple tandem transgene copies allows for labeling of individual neurons and glia in peripheral and central neurons with nuclear localized, membrane-targeted, or cytoplasmic fluorescent proteins in cre recombined cells.
Molecular note: The Thy1-Brainbow 2.1 transgene was designed with the mouse Thy1 regulatory elements surrounding four fluorescent proteins (green (GFP), enhanced yellow (EYFP), red (RFP), and cyan (CFP)) sequences uniquely positioned in a tandem fashion and delimited by LoxP sites in opposite orientation. Specifically, this Brainbow 2.1 coding region is composed of two adjacent head to tail tandem dimers, the first contains a LoxP site, humanized Renilla GFP (hrGFPII (Stratagene); with nuclear localization signal), and polyA sequence in forward orientation, and a LoxP site, monomeric EYFP (mYFPA206K), and polyA sequence in reverse orientation. The second head to tail dimer contains a LoxP site, tdimer2(12) RFP, and polyA sequence in forward orientation, and a LoxP site, mCerulean CFP (with membrane tethering palmitoylation sequence), and polyA sequence in reverse orientation. A single FRT site is located at the 3' end of the coding region. The hrGFPII variant of GFP (from Stratagene vector phrGFPII-C) has amino acid substitutions designed to improve spectral properties and performance in mammalian systems. The monomeric EYFP (mYFPA206K) has an amino acid substitution replacing a hydrophobic region with a positively charged residue designed to prevent dimerization. The tdimer2(12) RFP is a non-oligomerizing DsRed variant with a 12 residue linker fusing two copies of the protein (tandem dimer). The monomeric Cerulean (mCerulean) is a variant of ECFP (ECFPS72A/Y145A/H148D/A206K) with amino acid substitutions designed to improve spectral properties and prevent dimerization.