B6;129S4-Gt(ROSA)26Sor^{tm4(CAG-mOrange2,-EGFP,-mKate2)Zjh}/J

Stock number: 028583
Product name: RGBow reporter
Research areas: Neurobiology Research, Research Tools

Description

These RGBow reporter mice express one of three membrane-tethered fluorescent proteins in a random manner upon cre-mediated recombination; this allows labeling of individual cell types with distinguishable color variations in cre recombined cells.

Detailed description

RGBow reporter mice homozygous for this conditional fluorescent allele are viable and fertile. Mice possess three farnesylated fluorescent protein sequences uniquely flanked with pairs of incompatible Lox sites alternated to create mutually exclusive recombination events. This allows stochastic expression of multiple fluorescent proteins from a single locus. Prior to cre-mediated recombination, expression of the fluorescent proteins is restricted by a translational STOP sequence. When bred to Cre recombinase expressing mice, the resulting offspring can have one of three expression outcomes in each cell of the cre expressing tissue(s): mOrange2 (monomeric RFP), EGFP, or mKate2 (monomeric far-red RFP). A farnesylation sequence tethers the fluorophores to the membrane, allowing clear labeling of each cell.

Development

The RGBow targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), and three farnesylated fluorescent protein sequences uniquely flanked with pairs of incompatible Lox sites. Specifically, RGBow coding region contains (from 5' to 3') a LoxP site, a Lox2272 site, a LoxN site, a transcriptional STOP signal, mOrange2 sequence (derived from a monomeric red fluorescent protein) with polyA sequence, a LoxP site, EGFP (enhanced green fluorescent protein) with polyA sequence, a Lox2272 site, mKate2 (derived from a monomeric far-red fluorescent protein). This entire construct was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus via electroporation of (C57BL/6 x 129S4/SvJae)F1-derived V6.5 embryonic stem (ES) cells. Correctly targeted ES cells were selected and chimeric males were bred to C57BL/6J females and offspring were subsequently bred together to create a homozygous colony of RGBow reporter mice. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Gt(ROSA)26Sor^{tm4(CAG-mOrange2,-EGFP,-mKate2)Zjh}
Name: targeted mutation 4, Z Josh Huang
MGI accession ID: MGI:5700411
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: (C57BL/6 x 129S4/SvJae)F1
Expressed genes: GFP,Green Fluorescent Protein,; RFP,Red Fluorescent Protein,coral
Site of expression: One of either mOrange2 (monomeric RFP), EGFP, or mKate2 (monomeric far-red RFP) is expressed in cre-expressing cells after cre recombination.
Molecular note: The targeting vector is designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), and three farnesylated fluorescent protein sequences uniquely flanked with pairs of incompatible lox sites. Specifically, the coding region contains (from 5' to 3') a loxP site, a lox2272 site, a loxN site, a transcriptional STOP signal, mOrange2 sequence (derived from a monomeric red fluorescent protein) with polyA sequence, a loxP site, EGFP (enhanced green fluorescent protein) with polyA sequence, a lox2272 site, mKate2 (derived from a monomeric far-red fluorescent protein). This entire construct was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus.