B6.Cg-Igs7^{tm211(tetO-ChrimsonR/tdTomato,CAG-tTA2)Tasic}/J

Stock number: 037379
Product name: Ai211
Strain synonyms: TITL-ChrimsonR-tdT-Kv2.1-ICF-IRES-tTA2
Research areas: Cell Biology Research, Neurobiology Research, Research Tools

Description

Ai211 (also called Ai211(TITL-ChrimsonR-tdT-Kv2.1-ICF-IRES-tTA2)) is a dual-recombinase responsive (cre/flp-dependent), Tet-controllable, fluorescent and optogenetic tool allele created by targeted insertion at the Igs7 locus (TIGRE). Exposure to both Cre and Flp recombinase results in expression of tTA2 and ChrR-tdT (a light-gated cation channel; neuronal activation) in those cells where cre and flp expression overlaps/intersects. ChrR-tdT expression may be expected to be diminished/eliminated by doxycycline.

Detailed description

Ai211 mice harbor the TIGRE-Ins-tetO-LSL-ChrR-tdT-Ins-CAG-FSF-IRES-tTA2 conditional allele, designed with a tetO promoter, loxP-STOP-loxP cassette and ChrR-tdT sequence, and then a CAG promoter, FRT-STOP-FRT cassette and IRES-tTA2 sequence. ChrR-tdT is a light-gated cation channel that can be used as an optogenetic tool to activate neurons - see additional information below.

Prior to recombinase exposure, this allele is designed to have the two STOP cassettes preventing expression of the downstream genes (ChrR-tdT and tTA2). Ai211 heterozygotes are viable and fertile with no reported gross physical or behavioral abnormalities. To date (May 2022), it has not been attempted to make this strain homozygous.

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 overlaps - resulting in ChrR-tdT expression and tTA2 expression. ChrR-tdT is fused to a rat Kv2.1 potassium channel gene (Kcnb1) C-terminal sequence encoding a 65 amino acid sequence that functions to restrict protein localization to the soma and proximal dendrites.

Specifically, the donating investigator reports Ai211 exhibits no visible ChrR-tdT fluorescence in the absence of both cre and flp. Of note, the Ai211 allele was designed with an IRES upstream of the tTA2 to reduce/eliminate leakiness in the absence of Flp recombinase (Graybuck et al. 2021 Neuron [PMID:33789083]).

The donating investigator reports when Ai211 mice are bred with neocortical layer-specific and interneuron cre-driver and flp-driver lines to create multiple mutant animals, robust native ChrR-tdT expression in fixed brain tissue has been observed.

ChrR-tdT is the result of a ChrimsonR coupled to the fluorescent reporter tdTomato. ChrimsonR (ChrR) is a light-gated cation channel that can be used as an optogenetic tool to activate neurons. It is optimally excited by light of approximately 590nm wavelength (aka, red-shifted). ChrimsonR is an artificial channelrhodopsin derived by introducing the K176R mutation into the alga-derived channelrhodopsin Chrimson. The mutation results in faster off-kinetics without altering the action spectrum.

Development

The Ai211 allele (or Ai211(TITL-ChrimsonR-tdT-Kv2.1-ICF-IRES-tTA2)) has a Tet response element (tetO), loxP-STOP-loxP cassette and a ChrimsonR-tdTomato (ChrR-tdT) sequence that is followed by a CAG promoter, FRT-STOP-FRT cassette and IRES-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') a Bxb1 attL site, a partial green fluorescent protein sequence (non-functional 500bp fragment to increase the space between the Bxb1 attL 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 Tet response element/promoter (tetO), a loxP-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), ChrimsonR-tdTomato (ChrR-tdT) sequence with a Kv2.1 sequence at the 3' end followed by a 6xHis tag, T7 tag and XPress tag sequence, 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, a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG; from pCAGGS), a FRT-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-TKpA), an internal ribosome entry site (IRES) sequence (allows translation initiation in the middle of an mRNA sequence), a synthetic modified tetracycline-regulated transactivator gene (tTA2^S), a WPRE, a BGH polyA, a PhiC31 attB site, a PGK-5'hygro cassette, an mRNA 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 a mouse codon-optimized Bxb1 integrase recombinase vector for recombinase-mediated cassette exchange (RCME). Chimeric mice were bred with C57BL/6J mice. The Ai211 allele is therefore TIGRE-Bxb1 attL-partial GFP-Ins-tetO-LSL-ChrR-tdT-6xHis/T7/Xpress-WPRE-BGHpA-Ins-CAG-FSF-IRES-tTA2-WPRE-BGHpA-PhiC31 attB-PGK-5'hygro-SD-Bxb1 attR-SA-3'hygro-SV40pA-PhiC31 attP. The resulting Ai211 mice were bred with C57BL/6J for four generations. In 2022, males from generation N5 (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).

ChrR-tdT is the result of a ChrimsonR coupled to the fluorescent reporter tdTomato. ChrimsonR (ChrR) is a light-gated cation channel is optimally excited by light of approximately 590nm wavelength (aka, red-shifted). ChrimsonR is an artificial channelrhodopsin derived by introducing the K176R mutation into the alga-derived channelrhodopsin Chrimson.

Of note, the Ai211 allele was designed with an IRES upstream of the tTA2 to reduce/eliminate leakiness in the absence of Flp recombinase (Graybuck et al. 2021 Neuron [PMID:33789083]).

Allele

Symbol: Igs7^{tm211(tetO-ChrimsonR/tdTomato,CAG-tTA2)Tasic}
Name: targeted mutation 211, Bosiljka Tasic
MGI accession ID: MGI:7331584
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inducible; Transactivator; Epitope tag
Synonyms: Ai211; Ai211^{TITL-ChrimsonR-tdT-Kv2.1-ICF-IRES-tTA2}; Igs7^{tm211(tetO-ChrimsonR/tdTomato,CAG-tTA2)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 attL 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 Tet response element/promoter (tetO), a loxP-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), ChrimsonR-tdTomato (ChrR-tdT) sequence with a Kv2.1 sequence at the 3' end followed by a 6xHis tag, T7 tag and XPress tag sequence, 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, a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG; from pCAGGS), a FRT-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-TKpA), an internal ribosome entry site (IRES) sequence, a synthetic modified tetracycline-regulated transactivator gene (tTA2(S)), a WPRE, a BGH polyA, a PhiC31 attB site, a PGK-5'hygro cassette, an mRNA splice donor, a Bxb1 attR site, SA, 3' hygro-SV40pA, and PhiC31 attP. ChrR-tdT is the result of a ChrimsonR coupled to the fluorescent reporter tdTomato. ChrimsonR (ChrR) is a light-gated cation channel is optimally excited by light of approximately 590nm wavelength (aka, red-shifted). ChrimsonR is an artificial channelrhodopsin derived by introducing the K176R mutation into the alga-derived channelrhodopsin Chrimson.