B6.Cg-Igs7^{tm162.1(tetO-GCaMP6s,CAG-tTA2)Hze}/J

Stock number: 031562
Product name: Ai162(TIT2L-GC6s-ICL-tTA2)-D (or Ai162D)
Research areas: Cell Biology Research, Neurobiology Research, Research Tools

Description

Ai162(TIT2L-GC6s-ICL-tTA2)-D (also called Ai162D) mice are a Cre-dependent, Tet-controllable, fluorescent calcium-indicator tool strain - created by targeted insertion at the Igs7 locus (TIGRE; an intergenic region on mouse chromosome 9 that allows reporter expression to be tightly regulated). Exposure to Cre recombinase removes both STOP cassettes, resulting in expression of tTA2 and GCaMP6s (an ultrasensitive detector of single neuronal action potentials with slower decay and response kinetics). GCaMP6s expression may then be diminished by doxycycline.

Detailed description

Ai162(TIT2L-GC6s-ICL-tTA2)-D (also called Ai162D) mice harbor the TIGRE-Ins-TRE2-LSL-GCaMP6s-Ins-CAG-LSL-tTA2 conditional allele, designed with a TRE2 promoter, loxP-flanked STOP cassette and GCaMP6s sequence, and then a CAG promoter, lox2272-flanked STOP cassette and tTA2 sequence. Prior to Cre recombinase exposure, this allele is designed to have the two floxed-STOP cassettes preventing expression of the downstream genes (GCaMP6s and tTA2). When bred to mice expressing Cre recombinase, the offspring will have both floxed-STOP cassettes deleted in the cre-expressing cells/tissues - resulting in GCaMP6s expression and tTA2 expression. When GCaMP6s is expressed, subsequent calcium binding (such as neuronal activation) results in robust EGFP fluorescence. Further administration of tetracycline (or its analog doxycycline [dox]) will directly inhibit tTA2 activity, and therefore is expected to reduce GCaMP6s expression levels.

Specifically, the donating investigator reports Ai162D exhibit no native GCaMP6s fluorescence in the absence of Cre. When Ai162D mice are bred with Cre-driver lines to create double transgenic animals (Ai162D+/Cre+), cells expressing both tTA2 and Cre exhibit baseline GCaMP6s fluorescence in the absence of calcium binding. Following calcium binding (such as neuronal activation), a robust increase in GCaMP6s fluorescence is observed. To date (January 2018), Ai162D animals have not been characterized regarding dox-inducible attenuation of GCaMP6s expression, nor the Cre-inducible Ai162D expression in tissues other than brain.
For example, combining Ai162 with Slc17a7-IRES2-Cre (see Stock No. 023527) generates double transgenic Slc17a7-IRES2-Cre;Ai162 animals with strong GCaMP6s expression in pan-cortical glutamatergic neurons (and no aberrant [interictal] activity in animals tested to date [n=7; January 2018]). The same may be expected for Ai162D.
The donating investigator reports several examples where Ai162 crosses to neocortical layer-specific Cre-driver lines (e.g., Calb1-IRES2-Cre [see Stock No. 028532]) or interneuron Cre-driver lines (e.g., Sst-IRES2-Cre [see Stock No. 013044]) result in double transgenic mice with robust native GCaMP6s expression. The same may be expected for Ai162D.
Additionally, the donating investigator reports crossing Ai162 with the pan-excitatory Emx1-IRES-Cre line (Stock No. 005628) or the pan-inhibitory Gad2-IRES-Cre line (Stock No. 010802) may generate offspring with high levels of CAG-driven tTA2 expression early in development - which may result in reduced brain size and neurodegeneration and/or embryonic lethality, respectively. The same may be expected for Ai162D.

Ai162D heterozygotes are viable and fertile with no reported gross physical or behavioral abnormalities. In March 2026, the donating laboratory states that TIGRE knock-in alleles with long stretch of repetitive sequence and multiple transcription units could potentially lead to instability and unequal cross-over during meiosis. As such, the donating laboratory strongly recommends maintaining this strain as heterozygous.

The GCaMP6 slow variant calcium indicator (GCaMP6s) is an ultrasensitive detector of single neuronal action potentials with slower decay and response kinetics (Chen et al. 2013 Nature 499:295), and is an improved version of GCaMP5G. Compared to GCaMP6f, the slower kinetics render GCaMP6s more sensitive and slower to decay. 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.
The GCaMP6s fusion gene was designed by Drs. Douglas Kim and Loren Looger (Janelia Farm, HHMI). GCaMP6s has a chicken smooth muscle M13 fragment of myosin light chain kinase, a circularly permutated EGFP (cpEGFP; aa 149-238 followed by aa 1-144 [with M153K, V163A, S175G, D180Y, T203V, A206K and V93I mutations to increase dynamic range/baseline fluorescence, as well as K166H mutation for increased sensitivity/slower kinetics]), and a rat calmodulin DNA fragment (CaM; aa 2-148 with N60D, D78Y, T79R, S81T and R90G mutations [also called N362D, D380Y, T381R, S383T and R392G, respectively] to increase the fluorescence change for small calcium transients. The T302L and R303P amino acid substitutions in the cpEGFP-to-CaM linker improve sensor function.

Development

The Ai162(TIT2L-GC6s-ICL-tTA2)-D allele (or Ai162D) has a Tet response element TRE2, loxP-flanked-STOP cassette and GCaMP6s sequence followed by a CAG promoter, lox2272-flanked-STOP cassette and 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 pTRE2-LSL-GCaMP6s-CAG-LSL-tTA2 replacement vector was designed with (from 5' to 3') an frt3 site, 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 GCaMP6 slow variant calcium indicator sequence (GCaMP6s; details below), a 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 lox2272-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-TKpA), a synthetic modified tetracycline-regulated transactivator gene (tTA2^S), a WPRE, a BGH polyA, an AttB site, a PGK-5'hygro cassette, an RNA splice donor and a frt5 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 Flp recombinase vector for recombinase-mediated cassette exchange (RCME). Chimeras were bred with C57BL/6J-congenic PhiC31 mice (see Stock No. 007743) to remove the AttB/AttP-flanked PGK-hygro-SV40polyA cassette and replace it with the recombined AttB/AttP site (AttL). The Ai162D allele is therefore TIGRE-frt3-Ins-TRE2-LSL-GCaMP6s-WPRE-bGHpA-Ins-CAG-LSL-tTA2-WPRE-bGHpA-AttL. Resulting Ai162D mice were bred to C57BL/6J for several generations (and the PhiC31 gene was removed). 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 2018, heterozygous males from generation N7 (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).

Live colony rederive beginning November 2022 due to very low incidence of spontaneous coat color mutation:
In April 2022, one of our generation N7+pN3 breeding trios produced offspring with golden brindle stripes on right side of body, and therefore that breeding trio was discontinued. In October 2022, one of our generation N7+pN4F1 breeding trios produced one male offspring with brindle underside, and therefore that breeding trio was discontinued. The latter was not a descendent of the former, but they shared a common ancestor at generation N7+pN2. The genetic cause of this spontaneous coat color mutation was not determined. In November 2022, we will begin to rederive/refresh our entire live colony from sperm frozen in 2018 at generation N7. We will continue to breed black mice and will distribute black mice from breeding units that have not shown any unexpected coat colors.

TRE2 promoter information:
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.

GCaMP6s information:
The GCaMP6 slow variant calcium indicator (GCaMP6s) is an ultrasensitive detector of single neuronal action potentials with slow response kinetics (Chen et al. 2013 Nature 499:295), and is an improved version of GCaMP5G. The GCaMP6s fusion gene was designed by Drs. Douglas Kim and Loren Looger (Janelia Farm, HHMI). See strain description for more details on development and characteristics of GCaMP6s.

Allele

Symbol: Igs7^{tm162.1(tetO-GCaMP6s,CAG-tTA2)Hze}
Name: targeted mutation 162.1, Hongkui Zeng
MGI accession ID: MGI:6151062
Generation method: Targeted
Attributes: Reporter; Inducible
Synonyms: Ai162; Ai162(TIT2L-GC6s-ICL-tTA2)
Marker symbol: Igs7
Marker name: intergenic site 7
Marker synonyms: Iis7; TIGRE
Chromosome: 9
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: tTA,tetracycline-controlled transactivator,E. coli; GCaMP,Genetically encoded calcium indicator,
Site of expression: When bred to mice expressing Cre recombinase, the offspring will have both floxed-STOP cassettes deleted in the cre-expressing cells/tissues - resulting in GCaMP6s expression and tTA2 expression.
Molecular note: The vector is designed with (from 5' to 3') an FRT3 site, 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 GCaMP6 slow variant calcium indicator sequence (GCaMP6s), a 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 lox2272-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-TKpA), a synthetic modified tetracycline-regulated transactivator gene (tTA2^S), a WPRE, a BGH polyA, an AttB site, a PGK-5'hygro cassette, an RNA splice donor and a FRT5 site. 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. PhiC31-mediated recombination removed the AttB/AttP-flanked sequence and replaced it with the recombined AttB/AttP site (AttL).