NOD.129(Cg)-Gt(ROSA)26Sor^{tm4(ACTB-tdTomato,-EGFP)Luo}/YgchJ

Stock number: 037456
Product name: NOD.ROSA26-mTmG
Research areas: Neurobiology Research, Research Tools

Description

mT/mG (also called ROSA^mT/mG) is a cell membrane-targeted, two-color fluorescent Cre-reporter allele. Prior to Cre recombination, cell membrane-localized tdTomato (mT) fluorescence expression is widespread in cells/tissues. Cre recombinase expressing cells (and future cell lineages derived from these cells) have cell membrane-localized EGFP (mG) fluorescence expression replacing the red fluorescence.

In addition to this NOD/ShiLtJ congenic mT/mG strain (Stock No. 037456), the mT/mG reporter allele is also available on a C57BL/6J congenic background (Stock No. 007676) and a 129;CD1 mixed genetic background (Stock No. 007576).

The mT/mG reporter allele functions similarly to the ROSA^nT-nG reporter allele (nT/nG; Stock Nos. 023035/023537), with mT/mG directed to cell membrane and nT/nG directed to nuclei.

Detailed description

Mice homozygous for this mT/mG mutation are viable and fertile. These mice possess loxP sites on either side of a membrane-targeted tdTomato (mT) cassette and express strong red fluorescence in all tissues and cell types examined. Tail or whole body epifluorescence is sufficient to identify mT/mG mice from wildtype mice. When bred to cre recombinase expressing mice, the resulting offspring have the mT cassette deleted in the cre expressing tissue(s), allowing expression of the membrane-targeted EGFP (mG) cassette located just downstream. The donating investigator reports that the ACTB promoter allows stronger and persistent expression of the fluorescent proteins (especially in adult cells) compared to the endogenous Gt(ROSA)26Sor locus alone. This double-fluorescent system allows direct live visualization of both recombined and non-recombined cells at single cell resolution, offering an internal control for phenotypic analysis of Cre-induced mosaic mutants and providing a second marker for lineage tracing applications. In addition, the localization of fluorescent proteins to membrane structures outlines cell morphology and allows resolution of fine cellular processes. These mT/mG mice are useful as a Cre reporter strain; expressing red fluorescence prior to, and green fluorescence following, Cre-mediated recombination in widespread cell and tissue types.

Although it has not been determined if these mice develop autoimmune type 1 diabetes, the donating investigator suggests a footpad injection of Complete Freund's Adjuvant (CFA) administered once at weaning to delay diabetes onset, thus extending the lifespan of breeders.

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.

This allele is also available on a C57BL/6J congenic background as Stock No. 007676.

Development

The mT/mG (membrane-Tomato/membrane-Green) targeting vector was designed with a CMV enhancer/chicken beta-actin core promoter (pCA) driving expression of a loxP-flanked, N-terminal membrane-tagged, tdTomato protein sequence followed by a polyadenylation signal (tdTomato is a non-oligomerizing DsRed variant with a 12 residue linker fusing two copies of the protein (tandem dimer)). Immediately distal to the second loxP site is an N-terminal membrane-tagged, enhanced green fluorescent protein (EGFP) sequence itself followed by a polyadenylation signal. An frt-flanked neo cassette was also located distal to the expression vector. This entire mT/mG construct was inserted into the Gt(ROSA)26Sor locus via electroporation of (129X1/SvJ x 129S1/Sv)F1-derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were microinjected in C57BL/6J blastocysts. Chimeric progeny were bred to outbred CD-1 mice. The resulting mutant mice were interbred to generate homozygotes prior to arrival at The Jackson Laboratory (as Stock No. 007576). Upon arrival, some mice were backcrossed to C57BL/6J mice (Stock No. 000664) for at least five generations to generate a C57BL/6J congenic strain (as Stock No. 007676).

Dr. Yi-Guang Chen (Medical College of Wisconsin) obtained some of these animals and further backcrossed them to NOD/ShiLtJ mice (Stock No. 001976) for at least 11 generations before sending males back to The Jackson Laboratory. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize NOD/ShiLtJ oocytes.

Allele

Symbol: Gt(ROSA)26Sor^{tm4(ACTB-tdTomato,-EGFP)Luo}
Name: targeted mutation 4, Liqun Luo
MGI accession ID: MGI:3716464
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Synonyms: Gt(ROSA26)^{ACTB-tdTomato-EGFP}; mT/mG; mTmG; mTomato/mEGFP; R26^TG; R26-mR/mG; R26-X; ROSA^mT-mE; Rosa^mTmG; Rosa26^{flox-mTRed-Stop-flox-mGFP}; Rosa26^mTmG; ROSA26-mGFP; ROSA26-mTmG; Rosa26mTmG^flox; Rosa26R^mTmG; ROSA-mT/mG; ROSA-R/G; TomatoGFP+
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Expressed genes: RFP,Red Fluorescent Protein,coral; GFP,Green Fluorescent Protein,
Site of expression: Membrane-targeted tdTomato (mT) is expressed in all tissues and cell types examined. When bred to Cre recombinase expressing mice, the mT cassette is deleted in the cre expressing tissue(s), allowing expression of the membrane-targeted EGFP (mG).
Molecular note: The targeting vector was designed with a CMV enhancer/chicken beta-actin core promoter (pCA) driving expression of a loxP-flanked, N-terminal membrane-tagged, optimized DsRed fluorescent protein variant (called tandem-dimer-Tomato or tdTomato) sequence followed by a polyadenylation signal. Immediately distal to the second loxP site is an N-terminal membrane-tagged, enhanced green fluorescent protein (EGFP) sequence itself followed by a polyadenylation signal. An frt-flanked neo cassette was also located distal to the expression vector. Red fluorescence is detected in all tissues tested. When mice are bred to Cre expressing mice, the floxed region is excised in Cre-expressing tissue and this allows expression of the EGFP cassette.