B6;129S6-Gt(ROSA)26Sor^{tm8(CAG-mCherry,-EGFP)Dym}/J

Stock number: 029486
Product name: RC::FrePe dual-recombinase responsive fluorescent
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

The RC::FrePe dual-recombinase responsive fluorescent allele has a frt-flanked STOP and loxP-flanked mCherry::STOP all preventing transcription of eGFP. FLP recombinase results in mCherry fluorescence, and further exposure to Cre recombinase results in eGFP fluorescence in the overlapping populations. The RC::FrePe allele and its derivatives are useful for fluorescent labeling of cells/tissues in embryonic or adult mice. These mice allow intersectional genetic fate mapping of different cell subpopulations defined by the overlap of two gene expression domains.

Detailed description

The RC::FrePe dual-recombinase responsive fluorescent indicator allele has a frt-flanked STOP and loxP-flanked mCherry::STOP preventing transcription of an eGFP sequence. Although under control of the endogenous Gt(ROSA)26Sor promoter/enhancer regions and the CAG hybrid promoter, widespread expression of either fluorescent protein is prevented by STOP cassettes. After removal of the first STOP cassettes by FLP recombination, mCherry fluorescence is expected in Flp-expressing cells/tissues. Further exposure to Cre recombinase removes the floxed-tdTomato::STOP, resulting in eGFP fluorescence.

Mice homozygous for the RC::FrePe allele are viable and fertile with no reported gross physical or behavioral abnormalities.

FLP- and/or cre-mediated removal of specific STOP cassettes within the RC::FrePe allele results in the following derivative alleles:
i. FLP-mediated recombination results in the single-recombinase responsive indicator allele RC::rePe, which has constitutive mCherry fluorescence and allows cre-inducible expression of the eGFP.
ii. cre-mediated recombination results in the single-recombinase responsive indicator allele RC::Fe, which has no fluorescence (mCherry::STOP sequences removed) and allows FRT-inducible expression of eGFP.
iii. Combination of FLP- and cre-mediated recombination results in the RC::e allele (both STOPs deleted) - which has eGFP expression in the cells/tissues where FLP and cre expression overlap.

Development

The RC::FrePe dual-recombinase responsive fluorescent allele was created in the laboratory of Dr. Susan M. Dymecki (Harvard Medical School) to have a CAG promoter, a frt-flanked STOP, a loxP-flanked mCherry::STOP and an eGFP, all inserted into the Gt(ROSA)26Sor locus. The specific details are below.


The targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), a frt-flanked PGK-neo with transcriptional STOP cassette, a loxP site, a sequence encoding mCherry (an optimized DsRed/mRFP1 fluorescent protein variant with a combination of amino acid substitutions designed to improve spectral properties), a concatemer of SV40 polyA STOP sequences, a second loxP site, a sequence encoding enhanced green fluorescent protein (eGFP) and a polyA signal.

This entire targeting vector was inserted into the Gt(ROSA)26Sor locus via electroporation of embryonic stem (ES) cells. In 2016, RC::FrePe mice with black coat color were sent to The Jackson Laboratory Repository. Upon arrival, males were used to cryopreserve sperm. To establish the living RC::FrePe mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).

Allele

Symbol: Gt(ROSA)26Sor^{tm8(CAG-mCherry,-EGFP)Dym}
Name: targeted mutation 8, Susan Dymecki
MGI accession ID: MGI:5320888
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Synonyms: RC::FrePe; RC::rePe
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: Not Specified
Expressed genes: RFP,Red Fluorescent Protein,coral; GFP,Green Fluorescent Protein,
Site of expression: After removal of the first STOP cassettes by FLP recombination, mCherry fluorescence is expected in Flp-expressing serotonergic neurons. Further exposure to Cre recombinase removes the floxed-tdTomato::STOP, resulting in eGFP fluorescence.
Molecular note: The CAG promoter drives ubiquitous expression of an FRT-disrupted red mCherry and loxP-disrupted EGFP.