B6.Cg-Igs7^{tm210(tetO-GCaMP7f,CAG-tTA2)Tasic}/J

Stock number: 037378
Product name: Ai210
Strain synonyms: TITL-GC7f-ICF-IRES-tTA2
Research areas: Cell Biology Research, Neurobiology Research, Research Tools

Description

Ai210 (also called Ai210 (TITL-GC7f-ICF-IRES-tTA2)) is a dual-recombinase responsive (cre/flp-dependent), Tet-controllable, fluorescent calcium-indicator tool allele created by targeted insertion at the Igs7 locus (TIGRE). Exposure to both Cre and FLP recombinase results in expression of tTA2 and jGCaMP7f (an ultrasensitive EGFP fluorescent calcium detector of neuronal activity with faster decay and response kinetics) in those cells where cre and FLP expression overlaps/intersects. The jGCaMP7f expression may be expected to be diminished/eliminated by doxycycline. jGCaMP7f has high sensitivity and affinity, improved detection of individual spikes and may allow tracking of larger populations of neurons using two-photon imaging.

Detailed description

Ai210 mice harbor the TIGRE-Ins-tetO-LSL-jGCaMP7f-Ins-CAG-FSF-IRES-tTA2 conditional allele, designed with a tetO promoter, loxP-STOP-loxP cassette and jGCaMP7f sequence, and then a CAG promoter, FRT-STOP-FRT cassette and IRES-tTA2 sequence. jGCaMP7f is an ultrasensitive EGFP fluorescent calcium detector of neuronal activity with faster decay and response kinetics - see additional information below.

Prior to recombinase exposure, this allele is designed to have the two STOP cassettes preventing expression of the downstream genes (jGCaMP7f and tTA2). Ai210 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 jGCaMP7f expression and tTA2 expression. When jGCaMP7f is expressed, subsequent calcium binding (such as neuronal activation) may result in robust EGFP fluorescence. Further administration of tetracycline (or its analog doxycycline [dox]) will directly inhibit tTA2 activity, and therefore is expected to reduce jGCaMP7f expression levels.

Specifically, the donating investigator reports Ai210 exhibit no visible jGCaMP7f fluorescence in the absence of both cre and FLP. Of note, the Ai210 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 indicates that when Ai210 mice are bred with cre-driver and flp-driver lines to create multiple mutant animals, the Ai210+/cre+/flp+ cells exhibit moderate baseline jGCaMP7f fluorescence in the absence of calcium binding. The donating investigator reports that robust native GCaMP7f expression in fixed brain tissue has been observed in many triple transgenic mice with crosses of Ai210 to several neocortical layer-specific and interneuron driver lines (~10 total crosses). They have also analyzed GCaMP7f reporter fluorescence with in vivo calcium imaging in V1 and measured spontaneous and evoked responses. Also, they have delivered pan-neuronal AAVs to Ai210 mice and observed widespread native GCaMP7f expression.

To date (April 2021), Ai210 animals have not specifically been characterized regarding dox-inducible attenuation of jGCaMP7f expression, nor has the Ai210 mouse model been characterized for expression in tissues other than brain.

The "Janelia GCaMP7" sensitive and fast variant calcium indicator (jGCaMP7f) is an ultrasensitive detector of neuronal activity with faster decay and response kinetics (Dana et al. 2019 Nat Methods 16:649), and is an improved version of GCaMP6f. Compared to GCaMP6f, jGCaMP7f has a three-fold larger ΔF/F0 amplitude for one action potential stimuli and a faster rise time. In the absence of calcium binding, dim EGFP fluorescence is expected as there is a pore from the outside of its barrel into the chromophore. Upon calcium binding, this pore becomes occupied and fluorescence is increased.

Development

The Ai210 allele (or Ai210 (TITL-GC7f-ICF-IRES-tTA2)) has a Tet response element (tetO), loxP-STOP-loxP cassette and jGCaMP7f 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), a GCaMP7 sensitive and fast variant calcium indicator sequence (jGCaMP7f; details below) 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 Ai210 allele is therefore TIGRE-Bxb1 attL-partial GFP-Ins-tetO-LSL-jGCaMP7f -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 Ai210 mice were bred with C57BL/6J for five generations. In 2022, males from generation N6 (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).

The "Janelia GCaMP7" sensitive and fast variant calcium indicator (jGCaMP7f) is an ultrasensitive detector of neuronal activity with faster decay and response kinetics that was designed by Dr. Loren Looger (Janelia Farm, HHMI; Dana et al. 2019 Nat Methods 16:649). The jGCaMP7f fusion gene is an improved version of GCaMP6f. Like GCaMP6f, jGCaMP7f has a chicken smooth muscle M13 fragment of myosin light chain kinase (also called calmodulin-binding peptide [CBP]), a circularly permutated EGFP (cpEGFP; aa 149-238 followed by aa 1-144 [with mutations to increase dynamic range/baseline fluorescence, as well as increased sensitivity/slower kinetics]), and a rat calmodulin DNA fragment (CaM; aa 2-148 [with mutations to increase the fluorescence change for small calcium transients]). Amino acid substitutions in the cpEGFP-to-CaM linker improve sensor function. In addition, jGCaMP7f has the H78K mutation in GFP and the A317L, R381T, T383S, and G392R mutations in CaM.

Of note, the Ai210 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^{tm210(tetO-GCaMP7f,CAG-tTA2)Tasic}
Name: targeted mutation 210, Bosiljka Tasic
MGI accession ID: MGI:7331565
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inducible; Transactivator; Epitope tag
Synonyms: Ai210; Ai210 ^{TITL-GC7f-ICF-IRES-tTA2}; Igs7^{tm210(tetO-GCaMP7f,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), a GCaMP7 sensitive and fast variant calcium indicator sequence (jGCaMP7f; details below) 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. The "Janelia GCaMP7" sensitive and fast variant calcium indicator is an ultrasensitive detector of neuronal activity with faster decay and response kinetics. jThe allele carries a chicken smooth muscle M13 fragment of myosin light chain kinase (also called calmodulin-binding peptide [CBP]), a circularly permutated EGFP (cpEGFP; aa 149-238 followed by aa 1-144 [with mutations to increase dynamic range/baseline fluorescence, as well as increased sensitivity/slower kinetics]), and a rat calmodulin DNA fragment (CaM; aa 2-148 [with mutations to increase the fluorescence change for small calcium transients]). Amino acid substitutions in the cpEGFP-to-CaM linker improve sensor function. In addition, this allele has the H78K mutation in GFP and the A317L, R381T, T383S, and G392R mutations in CaM.