STOCK Gt(ROSA)26Sor^{tm7(CAG-dTomato,-EGFP)Zjh}/J

Stock number: 036760
Product name: IS2
Research areas: Neurobiology Research, Research Tools

Description

These IS2 reporter mice harbor a targeted mutation of the Gt(ROSA)26Sor locus with a loxP-flanked STOP cassette and an FRT-flanked dTomato (dimer Tomato) sequence preventing transcription of a CAG promoter-driven enhanced green fluorescent protein (EGFP). dTomato is expressed following cre-mediated recombination. EGFP is only expressed following cre-mediated and Flp-mediated recombination. This strain may be useful as an intersectional reporter for cre and FLP expression, or as a subtraction reporter for cre expression alone.

Detailed description

IS2 intersectional and subtraction 2 reporter mice homozygous for this Rosa-CAG-LSL-FRT-dTomato-FRT-EGFP conditional allele are viable and fertile. A loxP-flanked STOP cassette and an FRT-flanked dTomato cassette prevent transcription of the downstream EGFP sequence. Because this CAG promoter driven reporter construct was targeted for insertion into the Gt(ROSA)26Sor locus, dTomato expression is determined by which tissue(s) express Cre recombinase. dTomato (dimer Tomato) is a DsRed variant that is reported to be a somewhat slow-maturing dimer with low acid sensitivity. The dTomato sequence is half the length of that of tdTomato (tandem dimer Tomato), which facilitates recombination events. tdTomato has been designed for increased brightness and to facilitate efficient dimerization without the formation of monomer aggregates. In certain applications, for example, embryonic induction by Cre recombinase, the use of IS2 with the dTomato form leads to improved outcomes.

When bred to mice that express Cre recombinase, the resulting offspring will have the loxP-flanked STOP cassette deleted in the cre-expressing tissue(s). After cre excision, expression of EGFP is restricted by the FRT-flanked dTomato sequence. When bred to mice that express Flp recombinase, the resulting offspring will express EGFP only in cells that express Flp and previously expressed cre (intersectional), while still retaining dTomato expression in cre-expressing tissue(s) (subtraction).

IS mice are distributed as B6;129S4-Gt(ROSA)26Sor^{tm3(CAG-tdTomato,-EGFP*)Zjh}/J (Stock No. 028582), IS mice carry a tdTomato (tandem dimer) reporter rather than the dTomato (dimer) reporter carried by this strain.

Development

The IS2 targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), a loxP-flanked STOP cassette, an FRT-flanked dTomato sequence (dTomato is a DsRed variant that is reported to be a somewhat slow-maturing dimer with low acid sensitivity)), an EGFP, and a Neo-polyA cassette.This entire construct was inserted into 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 IS2 reporter mice (see SNP note below). 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).



In 2021, 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 males sent to The Jackson Laboratory Repository. Many of the 43 markers throughout the genome were segregating with an unknown source suggesting the mice sent to The Jackson Laboratory Repository were on a mixed genetic background.

Allele

Symbol: Gt(ROSA)26Sor^{tm7(CAG-dTomato,-EGFP)Zjh}
Name: targeted mutation 7, Z Josh Huang
MGI accession ID: MGI:6761516
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Synonyms: IS2
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: (C57BL/6 x 129S4/SvJae)F1
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), a loxP-flanked STOP cassette, an FRT-flanked dTomato (dimer Tomato) sequence (dTomato is a DsRed variant that is reported to be a somewhat slow-maturing dimer with low acid sensitivity)), an EGFP, and a Neo-polyA cassette.