B6.Cg-Igs7^{tm214(CAG-ACR1*,CAG-mRuby3)Tasic}/J

Stock number: 037380
Product name: Ai214
Strain synonyms: TICL-stGtACR1-HA-ICL-mRuby3
Research areas: Cell Biology Research, Neurobiology Research, Research Tools

Description

Ai214 (also called Ai214 (TICL-stGtACR1-HA-ICL-mRuby3)) is a Cre-dependent optogenetic and fluorescent tool allele designed with a CAG promoter, loxP-STOP-loxP and stGtACR1 coding sequence followed by a CAG promoter, lox2272-STOP-lox2272 and mRuby3 coding sequence - all inserted into the Igs7 locus (TIGRE). Exposure to Cre recombinase results in expression of stGtACR1 (soma-targeted variant of the red-shifted, light-gated anion channelrhodopsin GtACR1; neuronal silencing) and/or mRuby3 fluorescence.

Detailed description

Ai214 mice harbor the TIGRE-Ins-CAG-LSL-stGtACR1-Ins-CAG-FSF-mRuby3 conditional allele, designed with a CAG promoter, loxP-STOP-loxP cassette and stGtACR1 coding sequence, and then a CAG promoter, lox2272-STOP-lox2272 cassette and mRuby3 sequence.

Prior to Cre recombinase exposure, this allele is designed to have the two STOP cassettes preventing expression of the downstream genes (stGtACR1 and mRuby3). The donating investigator reports no background fluorescence is observed prior to removal of the STOP cassettes. Ai214 heterozygotes and homozygous mice are viable and fertile (Jan 2024).

When bred to mice expressing Cre recombinase, the offspring will have one or both STOP cassettes deleted in the cells where Cre is expressed - resulting in expression of stGtACR1 and/or mRuby3 fluorescence. stGtACR1 is a fusion of a soma trafficking domain from Kv2.1 (Kcnb1, potassium voltage gated channel, Shab-related subfamily, member 1) and GtACR1 (a red-shifted, light-gated anion channel rhodopsin derived from cryptophyte algae Guillardia theta). Anion channel rhodopsins (ACRs) provide highly sensitive and efficient membrane hyperpolarization and neuronal silencing through light-gated chloride conduction.

Specifically, the donating investigator reports when Ai214 mice are bred with Cre-driver lines to create double mutant animals, the Ai214+/Cre+ cells exhibit bright/robust fluorescence in the expected populations, including both stGtACR1 and mRuby3 fluorescence in any overlapping cell types.

The donating laboratory also notes that Ai214 mice exhibit no background levels of fluorescence in the absence of Cre.

To date (May 2022), Ai214 animals have not specifically been characterized for expression in tissues other than brain.

Development

The Ai214 allele (or Ai214 (TICL-stGtACR1-HA-ICL-mRuby3) has a CAG promoter, loxP-STOP-loxP cassette and stGtACR1 sequence that is followed by a CAG promoter, lox2272-STOP-lox2272 cassette and mRuby3 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 attL 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), the coding sequence of stGtACR1 (GtACR1 with targeting sequence TS and Kv2.1 and HA tag sequence), a BGH polyA, two copies of chicken beta-globin HS4 insulator element, the CAG promoter, a lox2272-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-TKpA), the coding sequence of mRuby3, 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 attL::PhiC31 attB-PGK/gb2-neoR-pA::Bxb1 attB::splice acceptor::3'hygro cassette::SV40pA::PhiC31 attR 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 Ai214 allele is therefore TIGRE-Bxb1 attL-partial GFP-Ins-CAG-LSL-stGtACR1-BGHpA-Ins-CAG-LSL-mRuby3-BGHpA-PhiC31 attB-PGK-5'hygro-SD-Bxb1 attR-SA-3'hygro-SV40pA-PhiC31 attP. The resulting Ai214 mice were bred with C57BL/6J for six generations. In 2022, males from generation N7 (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^{tm214(CAG-ACR1*,CAG-mRuby3)Tasic}
Name: targeted mutation 214, Bosiljka Tasic
MGI accession ID: MGI:7331587
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Synonyms: Ai214; Ai214 ^{TICL-stGtACR1-HA-ICL-mRuby3}; Igs7^{tm214(CAG-ACR1*,CAG-mRuby3)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 attL 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), the coding sequence of stGtACR1 (GtACR1 with targeting sequence TS and Kv2.1 and HA tag sequence), a BGH polyA, two copies of chicken beta-globin HS4 insulator element, the CAG promoter, a lox2272-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-TKpA), the coding sequence of mRuby3, 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. GtACR1 is a light-gated anion channel encoded by the Guillardia theta ACR1 gene.