B6.Cg-Igs7^{tm227(CAG-EGFP,CAG-tdTomato)Tasic}/J

Stock number: 037383
Product name: Ai227
Strain synonyms: TICF-nls-EGFP-ICF-tdT-WPRE
Research areas: Neurobiology Research, Research Tools

Description

Ai227 (also called Ai227 (TICF-nls-EGFP-ICF-tdT-WPRE)) is a FLP-recombinase reporter allele designed with a CAG promoter, ftr5-STOP-frt5 and EGFP coding sequence with a nuclear localization sequence followed by a CAG promoter, frt-STOP-frt and tdTomato coding sequence - all inserted into the Igs7 locus (TIGRE). Exposure to FLP recombinase results in nuclear localized EGFP fluorescence and/or nuclear localized tdTomato fluorescence.

Detailed description

Ai227 mice harbor the TIGRE-Ins-CAG-FSF-EGFP-Ins-CAG-FSF-tdT conditional allele, designed with a CAG promoter, frt5-STOP-frt5 cassette and a nuclear localized EGFP, and then a CAG promoter, frt-STOP-frt cassette and a tdTomato sequence.

Prior to recombinase exposure, this allele is designed to have the two STOP cassettes preventing expression of the downstream genes (nls EGFP and tdTomato). No background fluorescence is observed prior to removal of the STOP cassettes. Ai227 heterozygotes are viable and fertile with no reported gross physical or behavioral abnormalities. To date (June 2022), it has not been attempted to make this strain homozygous.

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

Specifically, the donating investigator reports robust native EGFP expression enriched in the nucleus in the brains of Ai227/wt;Flp/wt mice (~5 crosses) and with cell-type-specific AAVs delivered into Ai227/wt mice. The same has been observed for native tdTomato in the brains of Ai227/wt;FlpO/wt mice and with cell-type-specific AAVs.

To date (June 2022), Ai227 animals have not specifically been characterized for expression in tissues other than brain.

Development

The Ai227 allele (or Ai227 (TICF-nls-EGFP-ICF-tdT-WPRE) has a CAG promoter, frt5-STOP-frt5 cassette and EGFP sequence with a nuclear localization 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 frt5-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 tdTomato 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 Ai227 allele is therefore TIGRE-Bxb1 attL-partial GFP-Ins-CAG-FSF-nls EGFP-BGHpA-Ins-CAG-FSF-tdT-BGHpA-PhiC31 attB-PGK-5'hygro-SD-Bxb1 attR-SA-3'hygro-SV40pA-PhiC31 attP. The resulting Ai227 mice were bred with C57BL/6J for three generations. In 2022, males from generation N4 (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^{tm227(CAG-EGFP,CAG-tdTomato)Tasic}
Name: targeted mutation 227, Bosiljka Tasic
MGI accession ID: MGI:7331600
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Synonyms: Ai227; Ai227 (TICF-nls-EGFP-ICF-tdT-WPRE); Igs7^{tm227(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 (CAG; from pCAGGS), an FRT5-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 tdTomato 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.