B6;129S6-Igs7^{tm193(CAG-EGFP,CAG-tdTomato)Tasic}/J

Stock number: 034111
Product name: Ai193 [or Ai193(TICL-EGFP-ICF-tdT)]
Research areas: Neurobiology Research, Research Tools

Description

Ai193 (also called Ai193(TICL-EGFP-ICF-tdT)) is a dual-recombinase responsive reporter allele designed with a CAG promoter, loxP-STOP-loxP and EGFP coding sequence followed by a CAG promoter, frt-STOP-frt and tdTomato coding sequence - all inserted into the Igs7 locus (TIGRE). Exposure to either Cre or FLP recombinase results in EGFP fluorescence or tdTomato fluorescence, respectively. Furthermore, Ai193 are a Cre::FLP intersectional fluorescent reporter line as they are designed to allow both EGFP and tdTomato fluorescence in those cells where Cre and FLP expression overlaps.

Detailed description

Ai193 mice harbor the TIGRE-Ins-CAG-LSL-EGFP-Ins-CAG-FSF-tdT conditional allele, designed with a CAG promoter, loxP-STOP-loxP cassette and an EGFP, and then a CAG promoter, frt-STOP-frt cassette and tdTomato sequence.

Prior to recombinase exposure, this allele is designed to have the two STOP cassettes preventing expression of the downstream genes (EGFP and tdTomato). Some low levels of background fluorescence is observed prior to removal of the STOP cassettes, but those levels are very minimal compared to the fluorescence following recombinase exposure (as described below). Ai193 heterozygotes are viable and fertile with no reported gross physical or behavioral abnormalities. As of April 2021, it had not been attempted to make this strain homozygous. In March 2026, the donating laboratory states that, in attempting to make their Ai193 line homozygous, they observed one instance of a partial loss of the CAG promoter (potentially due to this TIGRE knock-in allele having a long stretch of repetitive sequence and multiple transcription units that could result in 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 EGFP fluorescence and tdTomato fluorescence.

Specifically, the donating investigator reports when Ai193 mice are bred with Cre-driver and FLP-driver lines to create triple mutant animals, the Ai193+/Cre+/FLP+ cells exhibit bright/robust fluorescence in the expected populations, including both EGFP and tdTomato fluorescence in any overlapping cell types. Examples of the Cre/FLP driver lines used for their characterization included VIP-IRES-Cre, Npnt-P2A-FlpO, Sncg-IRES2-FlpO-neo, Chrna6-IRES2-FlpO, Slc17a7-IRES2-FlpO, etc. Furthermore, PHP.eB serotyped viruses expressing Cre and FlpO delivered into Ai193 also resulted in the expected labeling.

To best ensure the Ai193 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 Ai193 reporter to generate triple mutant Ai193+/Cre+/FLP+ animals for their experiments.

The donating laboratory also notes that Ai193 exhibit low background levels of fluorescence in the absence of both Cre and FLP, but those levels are very minimal compared to the fluorescence following recombinase exposure. For example, when Ai193 are bred to Cre-driver mice, the resulting double mutant mice (Ai193+/Cre+/FLP-) had bright/robust EGFP fluorescence in the expected populations, with only very weak tdTomato expression. Conversely when Ai193 are bred to FLP-driver mice, the resulting double mutant mice (Ai193+/Cre-/FLP+) had bright/robust tdTomato fluorescence in the expected populations, with only very weak EGFP expression.

To date (April 2021), Ai193 animals have not specifically been characterized for expression in tissues other than brain.

Development

The Ai193 allele (or Ai193(TICL-EGFP-ICF-tdT) has a CAG promoter, loxP-STOP-loxP cassette and EGFP sequence that is followed by a CAG promoter, frt-STOP-frt cassette and tdTomato 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).

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), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), 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 tdTomato sequence (tdT; encoding the non-oligomerizing DsRed fluorescent protein variant with a 12 residue linker fusing two copies of the protein [tandem dimer]), a WPRE, a BGH polyA, a PhiC31 attB site, a PGK-5'hygro cassette, an RNA splice donor and a Bxb1 attB site. The development of this strain included Addgene Plasmid #181759.

(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 Ai193 allele is therefore TIGRE-Bxb1 attL-partial GFP-Ins-CAG-LSL-EGFP-WPRE-BGHpA-Ins-CAG-FSF-tdT-WPRE-BGHpA-PhiC31 attB-PGK-5'hygro-SD-Bxb1 attR-SA-3'hygro-SV40pA-PhiC31 attP. The resulting Ai193 mice were bred with C57BL/6J for several generations. In 2020, males from generation N3 (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^{tm193(CAG-EGFP,CAG-tdTomato)Tasic}
Name: targeted mutation 193, Bosiljka Tasic
MGI accession ID: MGI:6514381
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Synonyms: Ai193; Igs7^{tm193(CAG-EGFP,CAG-tdTomato)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 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, a loxP-flanked STOP cassette, a synthetic enhanced green fluorescent protein sequence (EGFP), a woodchuck hepatitis virus post-transcriptional regulatory element, 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 tdTomato sequence, a WPRE, a BGH polyA, a PhiC31 attB site, a PGK-5'hygro cassette, an RNA splice donor, Bxb1 attR-SA-3'hygro-SV40pA-PhiC31 attP.