B6;129S6-Igs7^{tm195(tetO-GCaMP7s,CAG-tTA2)Tasic}/J

Stock number: 034112
Product name: Ai195 [or Ai195(TIT2L-GCaMP7s-ICF-IRES-tTA2)]
Research areas: Cell Biology Research, Neurobiology Research, Research Tools

Description

Ai195 (also called Ai195(TIT2L-GCaMP7s-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 jGCaMP7s (an ultrasensitive EGFP fluorescent calcium detector of neuronal activity with slower decay and response kinetics) in those cells where Cre and FLP expression overlaps/intersects. The jGCaMP7s expression may be expected to be diminished/eliminated by doxycycline. jGCaMP7s has high sensitivity and affinity, improved detection of individual spikes and may allow tracking larger populations of neurons using two-photon imaging.

Detailed description

Ai195 mice harbor the TIGRE-Ins-TRE2-LSL-jGCaMP7s-Ins-CAG-FSF-IRES-tTA2 conditional allele, designed with a TRE2 promoter, loxP-STOP-loxP cassette and jGCaMP7s sequence, and then a CAG promoter, frt-STOP-frt cassette and IRES-tTA2 sequence. jGCaMP7s is an ultrasensitive EGFP fluorescent calcium detector of neuronal activity with slower 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 (jGCaMP7s and tTA2). Ai195 heterozygotes are viable and fertile with no reported gross physical or behavioral abnormalities. To date (February 2021), 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 overlap - resulting in jGCaMP7s expression and tTA2 expression. When jGCaMP7s 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 jGCaMP7s expression levels.

Specifically, the donating investigator reports Ai195 exhibit no visible jGCaMP7s fluorescence in the absence of both Cre and FLP. For example, crossing Ai195 to Gad2-IRES-Cre, Slc17a7-IRES2-Cre, or Snap25-IRES2-Cre resulted in no visible jGCaMP7s fluorescence. Furthermore, crossing Ai195 to Slc17a7-IRES2-FlpO-neo resulted in no visible jGCaMP7s fluorescence. Of note, the Ai195 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 Ai195 mice are bred with Cre-driver and FLP-driver lines to create multiple mutant animals, the Ai195+/Cre+/FLP+ cells exhibit moderate baseline jGCaMP7s fluorescence in the absence of calcium binding. Following calcium binding (such as neuronal activation), a significant increase in jGCaMP7s fluorescence is observed. Specifically, they observed increased fluorescence following spontaneous or evoked activity when imaging in the primary visual cortex of awake-behaving mice. In addition, they report Ai195;Gad2-IRES-Cre;Slc32a1-T2A-FlpO triple mutant cells demonstrate robust and bright jGCaMP7s expression.

To date (April 2021), Ai195 animals have not specifically been characterized regarding dox-inducible attenuation of jGCaMP7s expression, nor has the Ai195 mouse model been characterized for expression in tissues other than brain.

The "Janelia GCaMP7" sensitive and slow variant calcium indicator (jGCaMP7s) is an ultrasensitive detector of neuronal activity with slower decay and response kinetics (Dana et al. 2019 Nat Methods 16:649), and is an improved version of GCaMP6s. Compared to GCaMP6s, jGCaMP7s produces a high response to small (1-10) action potential trains - presenting a five-fold larger ΔF/F0 amplitude for one action potential stimuli and faster rise time. jGCaMP7s has a lower dissociation constant (higher affinity) for Ca2+ binding than GCaMP6s. 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 Ai195 allele (or Ai195(TIT2L-GCaMP7s-ICF-IRES-tTA2)) has a Tet response element TRE2, loxP-STOP-loxP cassette and jGCaMP7s 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 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 Tet response element/promoter (TRE2; details below), a loxP-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), a GCaMP7 sensitive and slow variant calcium indicator sequence (jGCaMP7s; 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 RNA splice donor and a Bxb1 attB 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 Ai195 allele is therefore TIGRE-Bxb1 attL-partial GFP-Ins-TRE2-LSL-jGCaMP7s-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 Ai195 mice were bred with C57BL/6J for several generations. In 2020, males from generation N3 (agouti 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 TRE2 promoter used here is Tet-responsive P_hCMV*-1; containing the Tet response element (seven copies of the 19 bp tet operator sequence [tetO]) just upstream of a minimal cytomegalovirus promoter (P_{min CMV}), which lacks the enhancer that is part of the complete CMV promoter. Consequently, P_hCMV*-1 is silent in the absence of tTA or rtTA binding to tetO.

The "Janelia GCaMP7" sensitive and slow variant calcium indicator (jGCaMP7s) is an ultrasensitive detector of neuronal activity with slower decay and response kinetics that was designed by Dr. Loren Looger (Janelia Farm, HHMI; Dana et al. 2019 Nat Methods 16:649). The jGCaMP7s fusion gene is an improved version of GCaMP6s. Like GCaMP6s, jGCaMP7s 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, jGCaMP7s has the A52V mutation in CBP and the A317L mutation in CaM.

Of note, the Ai195 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^{tm195(tetO-GCaMP7s,CAG-tTA2)Tasic}
Name: targeted mutation 195, Bosiljka Tasic
MGI accession ID: MGI:6514385
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inducible; Transactivator
Synonyms: Ai195; Igs7^{tm195(tetO-GCaMP7s,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 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 Tet response element/promoter (TRE2; details below), a loxP-flanked STOP cassette (stop codons in all thr ee reading frames linked to synthetic pA-hGHpA-PGKpA), a GCaMP7 sensitive and slow variant calcium indicator sequence (jGCaMP7s; details below) followed by a 6xHis tag, T7 tag and XPress tag sequence, a woodchuck hepatitis virus post-transcriptional regulatory element, a BGH polyA, two copies of chicken beta-globin HS4 insulator element, a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter , an 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 RNA splice donor,a Bxb1 attR-SA-3'hygro-SV40pA-PhiC31 attP. The TRE2 promoter used here is Tet-responsive P)hCMV*-1(; containing the Tet response element (seven copies of the 19 bp tet operator sequence [tetO]) just upstream of a minimal cytomegalovirus promoter (P)min CMV(), which lacks the enhancer that is part of the complete CMV promoter. Consequently, P)hCMV*-1( is silent in the absence of tTA or rtTA binding to tetO. The "Janelia GCaMP7" sensitive and slow variant calcium indicator (jGCaMP7s) is an ultrasensitive detector of neuronal activity with slower decay and response kinetics. The jGCaMP7s fusion gene is an improved version of GCaMP6s. Like GCaMP6s, jGCaMP7s 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, jGCaMP7s has the A52V mutation in CBP and the A317L mutation in CaM.