D2.Cg-Tg(Thy1-Brainbow1.1)MLich/SjJ

Stock number: 026855
Research areas: Neurobiology Research, Research Tools

Description

These Brainbow 1.1 (founder line M) mice allow mutually exclusive expression of three membrane-targeted fluorescent proteins in astrocytes (including glia) throughout the brain, providing unambiguous delineation of boundaries between adjacent astrocytes in cre recombined cells, and may also be useful in conjunction with other Brainbow strains (Stock No's. 007901, 007910, and 007921) for neurobiological studies.

Detailed description

These Thy1-Brainbow 1.1 (line M) transgenic mice are viable and fertile. The mice possess multiple fluorescent protein sequences uniquely flanked with pairs of incompatible Lox sites alternated to create mutually exclusive recombination events; allowing stochastic expression of multiple fluorescent proteins from a single transgene. Although the fluorescent protein immediately adjacent to the promoter, Kusabira-Orange (kOFP), was designed to be expressed prior to Cre-mediated recombination, basal kOFP expression is not observed in mouse tissues. When bred to Cre recombinase expressing mice, however, the resulting offspring can have one of three expression outcomes for each transgene in each cell of the cre expressing tissue(s): mCerulean (CFP), mYFP, or mCherry (RFP). The resulting fluorescent protein expression is observed in astrocytes of all areas of the brain and spinal cord, as well as dentate gyrus granule cells. A palmitoylation sequence tethers the mCherry (RFP), mYFP, and mCerulean (CFP) to the membrane, allowing clear labeling of axonal processes. 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 in the transgene allows tandem transgene copy number reduction through Flp-mediated recombination if desired. These Brainbow 1.1 (founder line M) mice allow mutually exclusive expression of three membrane-targeted fluorescent proteins in astrocytes (including glia) throughout the brain, providing unambiguous delineation of boundaries between adjacent astrocytes in cre recombined cells, and may also be useful in conjunction with other Brainbow strains (Stock No. 007901, 007910, and 007921) for neurobiological studies.

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

In an attempt to offer alleles on well-characterized or multiple genetic backgrounds, alleles are frequently moved to a genetic background different from that on which an allele was first characterized. It should be noted that the phenotype could vary from that originally described. We will modify the strain description if necessary as published results become available.

Development

The Thy1-Brainbow 1.1 transgene was designed with the mouse Thy1 regulatory elements surrounding four fluorescent proteins (orange (OFP), red (RFP), enhanced yellow (EYFP), and cyan (CFP)) sequences uniquely flanked with pairs of incompatible Lox sites. Specifically, this Brainbow 1.1 coding region contained (from 5’ to 3’) a LoxN site, Lox2272 site, LoxP site, Kusabira-Orange OFP (kOFP; with polyA sequence), LoxN site, mCherry RFP (with membrane tethering palmitoylation sequence and polyA sequence), Lox2272 site, monomeric EYFP (mYFP^A206K; with membrane tethering palmitoylation sequence and polyA sequence), LoxP site, mCerulean CFP (with membrane tethering palmitoylation sequence and 5’ FRT site and polyA sequence). This transgene was microinjected into C57BL/6JxCBA hybrid oocytes. Founder mice (line M) were bred with C57BL/6. The donating investigator reported that the resulting mutant mice were backcrossed to C57BL/6 (see SNP note below) 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.

Kusabira-Orange (KO or kOFP) is a true OFP with an artificial amino acid sequence attached to the N-terminus of the protein allowing bright orange fluorescence in cells. The mCherry RFP is an optimized DsRed/mRFP1 fluorescent protein variant with a combination of amino acid substitutions designed to improve spectral properties. The monomeric EYFP (mYFP^A206K) has an amino acid substitution replacing a hydrophobic region with a positively charged residue designed to prevent dimerization. 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.

A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. While the 27 markers throughout the genome suggested a C57BL/6 genetic background, 4 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6J ; C57BL/6N genetic background.

In this line, animals from Stock No. 007911 were backcrossed to DBA/2J for at least 10 generations by the donating laboratory.

Allele

Symbol: Tg(Thy1-Brainbow1.1)MLich
Name: transgene insertion M, Jeff W Lichtman
MGI accession ID: MGI:3761292
Generation method: Transgenic
Attributes: Reporter
Synonyms: Tg(Thy1-kOFP,-mCherry,-mYFP,-mCerulean)MLich; Thy1-brainbow-1.1 line M; Thy1-OFP
Marker symbol: Tg(Thy1-Brainbow1.1)MLich
Marker name: transgene insertion M, Jeff W Lichtman
Marker synonyms: Tg(Thy1-kOFP,-mCherry,-mYFP,-mCerulean)MLich; Thy1-brainbow-1.1 line M
Chromosome: UN
Strain of origin: C57BL/6 x CBA
Expressed genes: RFP,Red Fluorescent Protein,coral; YFP,Yellow Fluorescent Protein,jellyfish; OFP,Orange Fluorescent Protein,jelly fish; CFP,Cyan Fluorescent Protein,jellyfish
Site of expression: When bred to Cre recombinase expressing mice, there is one of three expression outcomes for each transgene copy in each cell of the cre expressing tissue(s): mCerulean (CFP), mYFP, or mCherry (RFP). Integration of multiple tandem transgene copies allows for labeling of individual astrocytes of all areas of the brain and spinal cord, as well as dentate gyrus granule cells.
Molecular note: The Thy1-Brainbow 1.1 transgene was designed with the mouse Thy1 regulatory elements surrounding four fluorescent proteins (orange (OFP), red (RFP), enhanced yellow (EYFP), and cyan (CFP)) sequences uniquely flanked with pairs of incompatible Lox sites. Specifically, this Brainbow 1.1 coding region contained (from 5' to 3') a LoxN site, Lox2272 site, LoxP site, Kusabira-Orange OFP (kOFP; with polyA sequence), LoxN site, mCherry RFP (with membrane tethering palmitoylation sequence and polyA sequence), Lox2272 site, monomeric EYFP (mYFPA206K; with membrane tethering palmitoylation sequence and polyA sequence), LoxP site, mCerulean CFP (with membrane tethering palmitoylation sequence and 5? FRT site and polyA sequence). Kusabira-Orange (KO or kOFP) is a true OFP with an artificial amino acid sequence attached to the N-terminus of the protein allowing bright orange fluorescence in cells. The mCherry RFP is an optimized DsRed/mRFP1 fluorescent protein variant with a combination of amino acid substitutions designed to improve spectral properties. The monomeric EYFP (mYFPA206K) has an amino acid substitution replacing a hydrophobic region with a positively charged residue designed to prevent dimerization. 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.