B6.Cg-Igs7^{tm148.1(tetO-GCaMP6f,CAG-tTA2)Hze}/J

Stock number: 030328
Product name: Ai148(TIT2L-GC6f-ICL-tTA2)-D (or Ai148D)
Research areas: Cell Biology Research, Neurobiology Research, Research Tools

Description

Ai148(TIT2L-GC6f-ICL-tTA2)-D (also called Ai148D) 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 GCaMP6f (a bright EGFP fluorescent calcium indicator of single neuronal action potentials with fast response kinetics). GCaMP6f expression may then be diminished/eliminated by doxycycline.

Detailed description

Ai148(TIT2L-GC6f-ICL-tTA2)-D (also called Ai148D) mice harbor the TIGRE-Ins-TRE2-LSL-GCaMP6f-Ins-CAG-LSL-tTA2 conditional allele, designed with a TRE2 promoter, loxP-flanked STOP cassette and GCaMP6f 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 (GCaMP6f and tTA2). When bred to mice expressing Cre recombinase, the resulting offspring will have both floxed-STOP cassettes deleted in the cre-expressing cells/tissues - resulting in GCaMP6f expression and tTA2 expression. When GCaMP6f is expressed, subsequent calcium binding (such as neuronal activation) results in robust EGFP fluorescence. Due to its placement downstream of the TRE2 promoter, subsequent introduction of tetracycline (or its analog doxycycline [dox]) will diminish/eliminate GCaMP6f expression.

Specifically, the donating investigator reports in the absence of Cre, mice heterozygous for Ai148D exhibit no fluorescence, and 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.
When Ai148D mice are bred with Cre-driver lines to create double transgenic animals (GCaMP6f+tTA2+/Cre+), cells expressing both tTA2 and Cre exhibit baseline EGFP fluorescence in the absence of calcium binding. Following calcium binding (such as neuronal activation), a robust increase in EGFP fluorescence is observed. The dox-inducible attenuation of EGFP expression has been characterized - EGFP fluorescence is completely suppressed after two weeks of dox treatment.
The donating investigator reports that double heterozygous Sst-IRES-Cre;Ai148D and VIP-IRES-Cre;Ai148D mice exhibit robust fluorescence with no viability problems (see Stock Nos. 013044, 010908). However, double heterozygous Emx1-IRES-Cre;Ai148D mice have reduced brain volume and pronounced neurodegeneration in all hippocampal subfields (see Stock No. 005628). In addition, their experience is double heterozygous Gad2-Cre;Ai148D mice exhibited embryonic lethality (see Stock No. 019022) - this is similar to their finding of double heterozygous lethality using the same Gad2-Cre driver with the Ai139D or Ai140D allele (Stock Nos. 030219 or 030220).

The donating investigator also reports that the fluorescent intensity observed in double heterozygous Ai148D mice (GCaMP6f+tTA2+/Cre+) is significantly greater than that of triple transgenic Ai93D mice (GCaMP6f+/tTA+/Cre+ ; see Stock Nos. 024103, 024108 , 005628).

To date (March 2017), the Cre-inducible Ai148D expression in tissues other than brain has not been characterized.

The GCaMP6 fast variant calcium indicator (GCaMP6f) is a detector of single neuronal action potentials with fast response kinetics (Chen et al. 2013 Nature 499:295), and is an improved version of GCaMP5G. Compared to GCaMP6s, GCaMP6f has faster response but is less sensitive and faster 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 GCaMP6f fusion gene was designed by Drs. Douglas Kim and Loren Looger (Janelia Farm, HHMI). GCaMP6f 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]), 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, as well the A15E (A317E) mutation for accelerated kinetics. The T302L and R303P amino acid substitutions in the cpEGFP-to-CaM linker improve sensor function.

Development

The Ai148(TIT2L-GC6f-ICL-tTA2)-D allele (or Ai148D) has a Tet response element TRE2, loxP-flanked-STOP cassette and GCaMP6f 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-GCaMP6f-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 fast variant calcium indicator sequence (GCaMP6f; 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 Ai148D allele is therefore TIGRE-frt3-Ins-TRE2-LSL-GCaMP6f-WPRE-bGHpA-Ins-CAG-LSL-tTA2-WPRE-bGHpA-AttL. Resulting Ai148D mice were bred to C57BL/6J for several generations (and the PhiC31 gene was removed). In 2017, males from generation N6 (black coat color) were sent to The Jackson Laboratory. Upon arrival, sperm is cryopreserved. To establish a live colony, an aliquot of frozen sperm is 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 GCaMP6 fast variant calcium indicator (GCaMP6f) is a detector of single neuronal action potentials with fast response kinetics (Chen et al. 2013 Nature 499:295). The GCaMP6f 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 GCaMP6f.

Allele

Symbol: Igs7^{tm148.1(tetO-GCaMP6f,CAG-tTA2)Hze}
Name: targeted mutation 148.1, Hongkui Zeng
MGI accession ID: MGI:5904003
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inducible; Transactivator
Synonyms: Ai148(TIT2L-GC6f-ICL-tTA2)-D; Ai148D
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: GCaMP,Genetically encoded calcium indicator,; tTA,tetracycline-controlled transactivator,E. coli
Site of expression: When these mice are bred with Cre-driver lines, baseline EGFP fluorescence is observed in the absence of calcium binding and robust fluorescence is observed after calcium binding. Upon administration of dox, EGFP fluorescence is eventually completely suppressed.
Molecular note: The replacement vector was designed with (from 5' to 3') an FRT3 site, two copies of chicken beta-globin HS4 insulator element, a Tet response element/promoter (TRE2), a loxP-flanked STOP cassette, a GCaMP6 fast variant calcium indicator sequence (GCaMP6f; details below), a WPRE, 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, a synthetic modified tetracycline-regulated transactivator gene (tTA2S), a WPRE, a BGH polyA, an AttB site, a PGK-5'hygro cassette, an RNA splice donor and a FRT5 site. 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). PhiC31-mediated recombination removed the AttB/AttP-flanked sequence (PGK-Neo-polyA::frt5::RNA splice acceptor::3'hygro-polyA) and replaced it with the recombined AttB/AttP site (AttL). The GCaMP6f fusion gene has a chicken smooth muscle M13 fragment of myosin light chain kinase, a circularly permutated EGFP, and a rat calmodulin DNA fragment. The GCaMP6 fast variant calcium indicator (GCaMP6f) is a detector of single neuronal action potentials with fast response kinetics, and is an improved version of GCaMP5G. Compared to GCaMP6s, GCaMP6f has faster response but is less sensitive and faster 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.