B6.Cg-Igs7^{tm224(CAG-EGFP,CAG-dTomato)Tasic}/J

Stock number: 037382
Product name: Ai224
Strain synonyms: TICL-nls-EGFP-ICF-nls-dTom
Research areas: Neurobiology Research, Research Tools

Description

Ai224 (also called Ai224 (TICL-nls-EGFP-ICF-nls-dTom)) is a dual-recombinase responsive reporter allele designed with a CAG promoter, loxP-STOP-loxP and EGFP coding sequence with a nuclear localization sequence followed by a CAG promoter, frt-STOP-frt and dTomato coding sequence with a nuclear localization sequence - all inserted into the Igs7 locus (TIGRE). Exposure to either Cre or FLP recombinase results in nuclear localized EGFP fluorescence or nuclear localized dTomato fluorescence, respectively. Furthermore, Ai224 are a Cre::FLP intersectional fluorescent reporter line as they are designed to allow both nuclear localized EGFP and nuclear localized dTomato fluorescence in those cells where Cre and FLP expression overlaps.

Detailed description

Ai224 mice harbor the TIGRE-Ins-CAG-LSL-EGFP-Ins-CAG-FSF-tdT conditional allele, designed with a CAG promoter, loxP-STOP-loxP cassette and a nuclear localized EGFP, and then a CAG promoter, frt-STOP-frt cassette and a nuclear localized dTomato sequence.

Prior to recombinase exposure, this allele is designed to have the two STOP cassettes preventing expression of the downstream genes (nls EGFP and nls dTomato). No background fluorescence is observed prior to removal of the STOP cassettes. As of May 2024, Ai224 homozygotes are viable and fertile with no reported gross physical or behavioral abnormalities. However in March 2026, the donating laboratory states that TIGRE knock-in alleles with long stretch of repetitive sequence and multiple transcription units could potentially lead to instability and unequal cross-over during meiosis. As such, the donating laboratory strongly recommends maintaining this strain as heterozygous.

When bred to mice expressing Cre recombinase and FLP recombinase, the offspring will have both STOP cassettes deleted in the cells where Cre and FLP expression overlap - resulting in robust/bright nuclear EGFP fluorescence and dTomato fluorescence.

Specifically, the donating investigator reports robust native EGFP expression enriched in the nucleus in the brains of Ai224/wt;Cre/wt mice (~5 crosses) and with cell-type-specific AAVs delivered into Ai224/wt mice. The same has been observed for native dTomato in the brains of Ai224/wt;FlpO/wt mice and with cell-type-specific AAVs. It is expected that triple mutant Ai224+/Cre+/FLP+ cells will exhibit bright/robust fluorescence in the expected populations, including both EGFP and dTomato fluorescence in the nucleus any overlapping cell types.

To best ensure the Ai224 STOP cassettes remain intact until the desired removal, the donating laboratory suggests that researchers consider first creating their desired double mutant Cre+/Flp+ mice, and then breed them to the Ai224 reporter to generate triple mutant Ai224+/Cre+/FLP+ animals for their experiments.

To date (June 2022), Ai224 animals have not specifically been characterized for expression in tissues other than brain.

Development

The Ai224 allele (or Ai224 (TICL-nls-EGFP-ICF-nls-dTom) has a CAG promoter, loxP-STOP-loxP cassette and EGFP sequence with a nuclear localization sequence that is followed by a CAG promoter, frt-STOP-frt cassette and dTomato sequence with a nuclear localization sequence - all inserted into the T1 TIGRE locus (an intergenic region on chromosome 9 [between AB124611 and Carm1] determined to have low basal transcriptional activity and allow high Cre and/or Tet inducibility). The development of this strain included Addgene Plasmid #181760.

Specifically, the replacement vector was designed with (from 5' to 3') a Bxb1 attB site, a partial green fluorescent protein sequence (non-functional 500bp fragment to increase the space between the Bxb1 attB site and the insulator sequence to improve stability of the targeting vector), two copies of chicken beta-globin HS4 insulator element (to reduce reporter gene expression in absence of transactivators), a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG; from pCAGGS), a loxP-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), a synthetic enhanced green fluorescent protein sequence (EGFP) with a nuclear localization sequence, a BGH polyA, two copies of chicken beta-globin HS4 insulator element, the CAG promoter, a frt-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-TKpA), a dTomato sequence with a nuclear localization sequence (tdT; encoding the non-oligomerizing DsRed fluorescent protein variant with a 12 residue linker fusing two copies of the protein [tandem dimer]), a BGH polyA, a PhiC31 attB site, a PGK-5'hygro cassette, an RNA splice donor and a Bxb1 attR site.

(129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem cells previously targeted with Bxb1 attP::PhiC31 attB-PGK/gb2-neoR-pA::Bxb1 attP::splice acceptor::3'hygro cassette::SV40pA::PhiC31 attP in TIGRE, were re-transfected with the replacement vector and Bxb1 integrase recombinase vector for recombinase-mediated cassette exchange (RCME). Chimeric mice were bred with C57BL/6J mice. The Ai224 allele is therefore TIGRE-Bxb1 attL-partial GFP-Ins-CAG-LSL-nls EGFP-BGHpA-Ins-CAG-FSF-nsl dT-BGHpA-PhiC31 attB-PGK-5'hygro-SD-Bxb1 attR-SA-3'hygro-SV40pA-PhiC31 attP. The resulting Ai224 mice were bred with C57BL/6J for five generations. In 2022, males from generation N6 (black coat color) were sent to The Jackson Laboratory. Upon arrival, males are used to cryopreserve sperm. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

The donating investigator reports that, at least once during backcrossing, a heterozygous female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin).

Allele

Symbol: Igs7^{tm224(CAG-EGFP,CAG-dTomato)Tasic}
Name: targeted mutation 224, Bosiljka Tasic
MGI accession ID: MGI:7331594
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Synonyms: Ai224; Ai224 (TICL-nls-EGFP-ICF-nls-dTom); Igs7^{tm224(CAG-EGFP,CAG-dTomato)Hze}
Marker symbol: Igs7
Marker name: intergenic site 7
Marker synonyms: Iis7; TIGRE
Chromosome: 9
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Molecular note: The targeting vector contains from (from 5' to 3') a Bxb1 attB site, a partial green fluorescent protein sequence (non-functional 500bp fragment to increase the space between the Bxb1 attB site and the insulator sequence to improve stability of the targeting vector), two copies of chicken beta-globin HS4 insulator element (to reduce reporter gene expression in absence of transactivators), a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG; from pCAGGS), a loxP-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), a synthetic enhanced green fluorescent protein sequence (EGFP) with a nuclear localization sequence, a BGH polyA, two copies of chicken beta-globin HS4 insulator element, the CAG promoter, an FRT-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-TKpA), a dTomato sequence with a nuclear localization sequence (tdT; encoding the non-oligomerizing DsRed fluorescent protein variant with a 12 residue linker fusing two copies of the protein [tandem dimer]), a BGH polyA, a PhiC31 attB site, a PGK-5'hygro cassette, an RNA splice donor, a Bxb1 attR site, SA, 3'hygro, SV40pA, and PhiC31 attP.