B6.Cg-Gt(ROSA)26Sor^{tm1(CAG-mNeonGreen/mRuby2)Mnz}/J

Stock number: 032068
Product name: Rosa26^LSL-INDIA
Research areas: Apoptosis Research, Immunology, Inflammation and Autoimmunity Research, Research Tools

Description

Cre-mediated excision of a loxP-flanked stop cassette enables tissue-specific expression of the INDIA apoptosis reporter in these Rosa26^LSL-INDIA mice. When aCasp3 (Casp3, caspase 3; a marker for apoptosis) cleaves a linker, the mNeonGreen and mRuby2 components of INDIA are separated, resulting in FRET loss and increased emission from mNeonGreen.

Detailed description

Rosa26^LSL-INDIA mice carry a FRET-based INDIcator of Apoptosis (INDIA) knocked into the mouse Gt(ROSA)26Sor locus. INDIA expression is blocked by a loxP-flanked STOP cassette. Cre-recombinase mediated excision of the floxed STOP enables tissue-specific expression of INDIA. This strain is especially useful in studies of apoptosis.

The INDIA reporter consists of an optimized fluorescence (Forster) resonance energy transfer (FRET) pair: N-terminally Myc-tagged mNeonGreen and mRuby2 linked by an 18 amino acid peptide containing an aCasp3-cleavage site (DEVDG). When aCasp3 (Casp3, caspase 3; a marker for apoptosis) cleaves the linker, mNeonGreen and mRuby2 are separated, resulting in FRET loss (FRET^-) and increased emission from mNeonGreen. Non-apoptotic cells retaining uncleaved mNeonGreen/mRuby2 (FRET⁺) demonstrate more yellow fluorescence indicating a lower ratio of mNeonGreen/FRET fluorescence emission.

Ubiquitous deletion of the floxed stop cassette via crosses with CMV-cre mice produced a strain with widespread INDIA apoptosis reporter capabilities - Rosa^INDIA (see Stock No. 032069).

Also of potential interest, Igk^INDIA mice (Stock No. 0032067) are particularly useful in studies of B cell apoptosis.

Development

A CAG promoter, loxP-flanked STOP/neomycin cassette in reverse transcriptional orientation, a FRET-based INDIcator of Apoptosis (INDIA) cassette, a single FRT site (remaining after Flp-expressing bacteria were used to excise an FRT-flanked IRES-EGFP cassette from the targeting vector), and polyA were introduced to intron 1 of the mouse Gt(ROSA)26Sor gene via homologous recombination in B6(Cg)-Tyr^c-2J/J-derived CY2.4 embryonic stem (ES) cells. The resulting male chimeric animals were crossed to B6(Cg)-Tyr^c-2J/J females (see Stock No. 000058) to allow for coat color screening of germline transmission.

The INDIA reporter consists of an optimized fluorescence (Forster) resonance energy transfer (FRET) pair: N-terminally Myc-tagged mNeonGreen and mRuby2 linked by an 18 amino acid peptide containing an aCasp3-cleavage site (DEVDG). When aCasp3 (Casp3, caspase 3; a marker for apoptosis) cleaves the linker, mNeonGreen and mRuby2 are separated, resulting in FRET loss (FRET^-) and increased emission from mNeonGreen. Non-apoptotic cells retaining uncleaved mNeonGreen/mRuby2 (FRET⁺) demonstrate more yellow fluorescence indicating a lower ratio of mNeonGreen/FRET fluorescence emission.

This strain was maintained on an albino C57BL/6 genetic background by the donating laboratory (see SNP note below). The Tyr^c-2J allele was bred out of the colony at The Jackson Laboratory.

In 2020, a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 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. One of the 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/6N;C57BL/6J genetic background.

Allele

Symbol: Gt(ROSA)26Sor^{tm1(CAG-mNeonGreen/mRuby2)Mnz}
Name: targeted mutation 1, Michel C Nussenzweig
MGI accession ID: MGI:6156806
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Synonyms: Rosa26^LSL-INDIA
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: B6(Cg)-Tyr^c-2J
Expressed genes: RFP,Red Fluorescent Protein,coral; GFP,Green Fluorescent Protein,
Site of expression: Cre-mediated excision of a loxP-flanked stop cassette enables tissue-specific expression of the INDIA apoptosis reporter in these Rosa26^LSL-INDIA mice.
Molecular note: A CAG promoter, loxP-flanked STOP/neomycin cassette in reverse transcriptional orientation, a FRET-based INDIcator of Apoptosis (INDIA) cassette, a single FRT site (remaining after Flp-expressing bacteria were used to excise an FRT-flanked IRES-EGFP cassette from the targeting vector), and polyA were introduced to intron 1. The INDIA reporter consists of an optimized fluorescence (Forster) resonance energy transfer (FRET) pair: N-terminally Myc-tagged mNeonGreen and mRuby2 linked by an 18 amino acid peptide containing an aCasp3-cleavage site (DEVDG). When aCasp3 (Casp3, caspase 3; a marker for apoptosis) cleaves the linker, mNeonGreen and mRuby2 are separated, resulting in FRET loss (FRET<->) and increased emission from mNeonGreen. Non-apoptotic cells retaining uncleaved mNeonGreen/mRuby2 (FRET<+>) demonstrate more yellow fluorescence indicating a lower ratio of mNeonGreen/FRET fluorescence emission.