B6;129S6-Gt(ROSA)26Sor^{tm1(CAG-tdTomato*,-EGFP*)Ees}/J

Stock number: 023035
Product name: ROSA^nT-nG
Strain synonyms: nT/nG , nTnG
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

ROSA^nT-nG mice are available on a congenic C57BL/6NJ background (Stock No. 023537) and the congenic version will replace this mixed background strain in the near future.

ROSA^nT-nG (also called nT/nG) is a nuclear-targeted, two-color fluorescent Cre reporter allele that express nuclear-localized red fluorescence in a widespread fashion prior to Cre recombinase exposure. Following exposure to Cre, nuclear-localized green fluorescence is observed.

In addition to this B6;129 mixed genetic background ROSA^nT-nG strain (Stock No. 023035), the ROSA^nT-nG reporter allele is also available on a C57BL/6NJ congenic background (Stock No. 023537).

The ROSA^nT-nG reporter allele functions similarly to the mT/mG reporter allele (ROSA^mT/mG; Stock Nos. 007676/037456/007576), with nT/nG directed to nuclei and mT/mG directed to cell membrane.

Detailed description

The ROSA^nT-nG allele consists of a CMV enhancer/chicken beta-actin core promoter, a loxP-flanked nT cassette, and a nG cassette, all inserted into the Gt(ROSA)26Sor locus. Prior to exposure to Cre recombinase, strong red fluorescence is observed in the nuclei of cells/tissues in a widespread fashion. No EGFP expression is reported prior to cre-mediated recombination. Superficially, ROSA^nT-nG mice may be distinguished from wildtype by tail or whole body epifluorescence (tdTomato fluorescence). The red fluorescence occurs in bands around the tail (reflecting the distribution of the nuclei). When bred to Cre recombinase expressing mice, offspring will have the floxed nT cassette deleted in the cre expressing cells/tissues; this allows expression of the downstream nG cassette and results in strong green fluorescence in the nuclei of cre expressing cells and future cell lineages derived from these cells. This double-fluorescent system allows direct live visualization of both Cre 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 cell lineage tracing applications. In addition, localization of fluorescent proteins to nuclei is well-suited for examination of proliferating differentiated cell types (e.g., hepatocytes), tissues refractory to whole-cell flow cytometry analyses (e.g., liver, muscle, brain, kidney) and tissues with ploidy transitions (e.g., cancers, trophectoderm derivatives), as well as immuno-co-localization studies for cell type-specific transcription factors. The C-terminal SRm₁₆₀ domain effectively targets the fluorescent protein to the nucleus. While slow diffusion out of isolated nuclei is observed at room temperature in less than one hour, the use of cold temperatures, viscous buffers or mild fixation allows longer/permanent nuclear retention. Mice homozygous for the ROSA^nT-nG allele are viable and fertile.

Development

Dr. Edward E. Schmidt (Montana State University) created the nT-nG targeting vector by modifying the Rosa26 mT/mG plasmid (see Stock No. 007676) designed by Dr. Liqun Luo (Stanford University). Dr. Schmidt's modifications replaced the N-terminal membrane-targeting domains of tdTomato and EGFP with strong start codons, and added sequences at the C-termini of tdTomato and EGFP that encode amino acids 300-350 of the SRm₁₆₀ nuclear assembly domain (to direct the stable integration of each fluorescent protein into the nucleus).

The final nT-nG targeting vector contained (from 5' to 3') a CMV enhancer/chicken beta-actin core promoter (pCA), a loxP site, the nT cassette (tdTomato protein sequence [non-oligomerizing DsRed variant with a 12 residue linker fusing two copies of the protein (tandem dimer)] with C-terminal SRm₁₆₀ nuclear targeting signal and a polyadenylation signal), a second loxP site, the nG cassette (an enhanced green fluorescent protein [EGFP] sequence with C-terminal SRm₁₆₀ nuclear targeting signal and a polyadenylation signal), and an frt-flanked neo cassette. The SRm₁₆₀ sequences attached to tdTomato and EGFP contain different synonymous mutations (and the SRm₁₆₀ sequence in tdTomato also has a single non-synonymous mutation) to avoid potential homologous recombination in bacteria between the SRm₁₆₀ sequences in the nT cassette and nG cassette. The donating investigator reports that the nT-nG targeting construct was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus via electroporation into (129S6/SvEvTac x C57BL/6NCr)F1-derived G4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts, and chimeric males were bred with C57BL/6 females (substrain unknown) to generate founder animals. A male founder mouse (with the ROSA^nT-nG targeted mutation inserted on the 129-donor chromosome) was bred to C57BL/6NCrl females to establish the ROSA^nT-nG mutant mouse line. ROSA^nT-nG mice were then bred together for several generations prior to sending homozygous males to The Jackson Laboratory Repository in 2013. Upon arrival, they were bred to C57BL/6NJ inbred mice (Stock No. 005304) for at least one generation.

Allele

Symbol: Gt(ROSA)26Sor^{tm1(CAG-tdTomato*,-EGFP*)Ees}
Name: targeted mutation 1, Edward E Schmidt
MGI accession ID: MGI:5504463
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: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: GFP,Green Fluorescent Protein,; RFP,Red Fluorescent Protein,coral
Site of expression: Red fluorescence is observed in the nuclei of cells/tissues in a widespread fashion in mice carrying this allele prior to cre-mediated recombination.
Molecular note: The nT-nG targeting vector was made by modifying the Rosa26 mT/mG plasmid (see JAX Stock No. 007676). Modifications replaced the N-terminal membrane-targeting domains of tdTomato and EGFP with strong start codons, and added sequences at the C-termini of tdTomato and EGFP that encode amino acids 300-350 of the SRm160 nuclear assembly domain (to direct the stable integration of each fluorescent protein into the nucleus). The final nT-nG targeting vector contained (from 5' to 3') a CMV enhancer/chicken beta-actin core promoter (pCA), a loxP site, the nT cassette (tdTomato protein sequence [non-oligomerizing DsRed variant with a 12 residue linker fusing two copies of the protein (tandem dimer)] with C-terminal SRm160 nuclear targeting signal and a polyadenylation signal), a second loxP site, the nG cassette (an enhanced green fluorescent protein [EGFP] sequence with C-terminal SRm160 nuclear targeting signal and a polyadenylation signal), and an frt-flanked neo cassette. The SRm160 sequences attached to tdTomato and EGFP contain different synonymous mutations (and the SRm160 sequence in tdTomato also has a single non-synonymous mutation) to avoid potential homologous recombination in bacteria between the SRm160 sequences in the nT cassette and nG cassette.