B6.Cg-Igs7^{tm223(tetO-DhaA*,CAG-mScarlet,-tTA2)Hze}/J

Stock number: 039736
Product name: Ai223
Research areas: Neurobiology Research, Research Tools

Description

These Ai223 mice have a Tet response element (tetO), loxP-STOP-loxP cassette and Halotag sequence, and then a CAG promoter, frt-STOP-frt cassette and H2B-mScarlet-P2A-tTA2 sequence all inserted into the T1 TIGRE locus (Igs7). These mice are an intersectional line where exposure to both Cre and FLP recombinase results in expression of Halotag and H2B-mScarlet-P2A-tTA2 in those cells where Cre and FLP expression overlaps/intersects.

Detailed description

Ai223(TITL-HaloTag-ICF-mScarlet-H2B-P2A-tTA2) (also called Ai223) mice harbor the TIGRE-Ins-TRE-LSL-Halotag-Ins-CAG-FSF-mScarlet-H2B-P2A-tTA2 conditional allele, designed with a TRE promoter, loxP-STOP-loxP cassette and Halotag sequence, and then a CAG promoter, frt-STOP-frt cassette and H2B-mScarlet-P2A-tTA2 sequence. Prior to Cre and FLP recombinase exposure, this allele is designed to have the two STOP cassettes preventing expression of the downstream genes (Halotag, mScarlet, and tTA2).

When bred to mice expressing Cre recombinase, the offspring will have the floxed-STOP cassette deleted in the Cre-expressing cells/tissues - resulting in Halotag expression. HaloTag is a 33 kDa haloalkane dehalogenase encoded by the DhaA gene from Rhodococcus rhodochrous that has been mutagenized to form an irreversible covalent bond to synthetic chloroalkane ligands.

When bred to mice expressing FLP recombinase, the offspring will have the frt-flanked-STOP cassette deleted in the Flp-expressing cells/tissues - resulting in mScarlet and tTA2 expression.

Specifically, the donating investigator reports Ai223 exhibit no native fluorescence or reporter gene expression in the absence of Cre. Ai223 heterozygotes are viable and fertile with no reported gross physical or behavioral abnormalities. To date (August 2024), it has not been attempted to make this strain homozygous.

Development

Ai223(TITL-HaloTag-ICF-mScarlet-H2B-P2A-tTA2) (also called Ai223) mice have a a modified Tet response element (TRE or (tetO); described in detail below), loxP-flanked STOP cassette and Halotag sequence, and then a CAG promoter, frt-flanked STOP cassette and H2B-mScarlet-P2A-tTA2 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') an 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 the chicken beta-globin HS4 insulator element, a Tet response element/promoter (tetO), a loxP-STOP-loxP cassette, a farnesylated interchangeable ligand fusion protein (HaloTag; derived from Promega technologies), a woodchuck post-transcriptional regulatory element to increase mRNA stability (WPRE), a bovine growth hormone polyA (bGHpA), two copies of the chicken beta-globin HS4 insulator element, a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), an frt-flanked STOP cassette, a red fluorescent protein (mScarlet), a histone 2B (H2B), porcine teschovirus-1 2A (P2A), self-cleaving peptide, modified C22 tetracycline transactivator version 2 (tTA2), another WPRE sequence, another bGHpA sequence, PhiC31 AttB site, phosphoglycerate kinase 1 promoter (PGK), one domain of the hygromycin resistance gene, a splice domain and a mRNA splice acceptor sequence, a second domain of the hygromycin resistance gene, a SV40 pA sequence, and a PhiC31 AttP site.

(129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem cells previously targeted with frt3::AttB::PGK-neoR-polyA::frt5::splice acceptor::3'hygro cassette::SV40 polyA:AttP in TIGRE, were re-transfected with the replacement vector and mouse codon-optimized Bxb1 integrase recombinase vector for recombinase-mediated cassette exchange (RCME). The Ai223 allele is therefore TIGRE-Bxb1 attL-partial GFP-Ins-TRE2-LSL-Halotag-WPRE-BGHpA-Ins-CAG-FSF-H2B-mScarlet-P2A-tTA2-WPRE-BGHpA-PhiC31 attB-PGK-5'hygro-SD-Bxb1 attR-SA-3'hygro-SV40pA-PhiC31 attP. Resulting Ai223 mice were bred to C57BL/6J for several generations. 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). In 2024, heterozygous males from generation N5 (black coat color) were sent to The Jackson Laboratory. Upon arrival, sperm was cryopreserved. To establish a live colony, an aliquot of frozen sperm is used to fertilize C57BL/6J oocytes (Stock No. 000664).

The TRE promoter used here is Tet-responsive P_tight; containing a modified Tet response element (TRE_mod) composed of seven direct 36 bp repeats (each containing the 19 bp tet operator sequence [tetO]). TRE_mod is just upstream of a minimal human cytomegalovirus promoter (P_minCMVΔ), which lacks the enhancer that is part of the complete CMV promoter. Consequently, P_tight is silent in the absence of TetR or rTetR binding to tetO.

Allele

Symbol: Igs7^{tm223(tetO-DhaA*,CAG-mScarlet,-tTA2)Hze}
Name: targeted mutation 223, Hongkui Zeng
MGI accession ID: MGI:7712098
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inducible; Inserted expressed sequence; RMCE-ready; Transactivator; Epitope tag
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 replacement vector designed with (from 5' to 3') an 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 the chicken beta-globin HS4 insulator element, a Tet response element/promoter (tetO), a loxP-STOP-loxP cassette, a farnesylated interchangeable ligand fusion protein (HaloTag; derived from Promega technologies) a woodchuck post-transcriptional regulatory element to increase mRNA stability (WPRE), a bovine growth hormone polyA (bGHpA), two copies of the chicken beta-globin HS4 insulator element, a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), an frt-flanked STOP cassette, a red fluorescent protein (mScarlet), a histone 2B (H2B), porcine teschovirus-1 2A (P2A), self-cleaving peptide, modified C22 tetracycline transactivator version 2 (tTA2), another WPRE sequence, another bGHpA sequence, PhiC31 AttB site, phosphoglycerate kinase 1 promoter (PGK), one domain of the hygromycin resistance gene, a splice domain and a mRNA splice acceptor sequence, a second domain of the hygromycin resistance gene, a SV40 pA sequence, and a PhiC31 AttP site was 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).