STOCK Igs7^{tm93.1(tetO-GCaMP6f)Hze} Tg(Camk2a-tTA)1Mmay/J

Stock number: 024108
Product name: Ai93(TITL-GCaMP6f)-D;CaMK2a-tTA
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

Ai93(TITL-GCaMP6f)-D;CaMK2a-tTA (or Ai93D;CaMK2a-tTA) mice have a Cre/Tet-dependent, fluorescent calcium indicator GCaMP6f inserted into the Igs7 locus (TIGRE; an intergenic region on mouse chromosome 9 that allows reporter expression to be tightly regulated), as well as a transgene directing tetracycline-controlled transactivator protein (tTA) expression in forebrain neurons. After removal of the floxed-STOP cassette by Cre recombinase, resulting mice have doxycycline-inducible/reversible expression of GCaMP6 fast variant calcium indicator (GCaMP6f; a detector of single neuronal action potentials with fast response kinetics) in forebrain neurons. Following subsequent calcium binding (such as neuronal activation), bright EGFP fluorescence is observed.

As described for Stock No. 007004, the CaMK2a-tTA transgene integrated into chromosome 12 causing a 508 Kb deletion that spans the 3' half of Vipr2, the entire Wdr60, Esyt2, D430020J02Rik and Ncapg2 loci and the first two exons of Ptprn2. Homozygous mice will therefore have a functional knock-out of the deleted loci, and altered or null expression of Vipr2 and Ptprn2. Founder line 1 has >20 transgene copies [Goodwin et al. 2019 Genome Res. 29:494].

Detailed description

Ai93(TITL-GCaMP6f)-D;CaMK2a-tTA (or Ai93D;CaMK2a-tTA) mice have a Cre/Tet-dependent, fluorescent calcium indicator (GCaMP6f; a detector of single neuronal action potentials with fast response kinetics) inserted into the Igs7 locus (TIGRE; an intergenic region on mouse chromosome 9 that allows reporter expression to be tightly regulated), as well as a transgene directing tetracycline-controlled transactivator protein (tTA) expression in forebrain neurons.

In the absence of Cre recombinase, Ai93D;CaMK2a-tTA mice are designed to have tTA expression in forebrain neurons but GCaMP6f expression (EGFP fluorescence) is blocked by a floxed STOP cassette. Following exposure to Cre recombinase, the floxed STOP cassette in Ai93D is deleted; leading to expression of GCaMP6f in forebrain neurons. GCaMP6f expression can be regulated with tetracycline or its analog doxycycline (dox).

When Ai93D;CaMK2a-tTA mice are bred with Cre-driver lines to create triple transgenic animals (GCaMP6f+/tTA+/Cre+), EGFP fluorescence in cre-expressing cells is low in the absence of calcium binding. Following calcium binding (such as neuronal activation), bright EGFP fluorescence is observed in cells expressing Cre recombinase. See further phenotype considerations below.

The donating investigator also reports that following neuronal activation, the fluorescent intensity observed in triple transgenic Ai93D mice (GCaMP6f+/tTA+/Cre+) is significantly greater than that of the double transgenic Ai95D system (GCaMP6f+/Cre+ ; see Stock No. 024105). Although both systems utilize the same GCaMP6f gene, they differ in chromosomal location and Tet-inducibility.

Ai93D;CaMK2a-tTA mice heterozygous for both targeted mutations are viable and fertile with no reported gross physical or behavioral abnormalities. The phenotype of mice homozygous for both Ai93D and CaMK2a-tTA has not been characterized to date (April 2014).

Further phenotype considerations: Steinmetz et al. 2017 ENeuro reports aberrant neural activity resembling interictal spikes were observed in some genotypes of GCaMP6f-expressing Ai93 mice. This activity was most prominent in Emx1-Cre;Camk2a-tTA;Ai93 mice and Emx1-Cre;Rosa26-ZtTA;Ai93 mice, and also in Camk2a-tTA;Ai93 mice that had undergone germline Cre recombination. Aberrant neural activity was also occasionally observed in Slc17a7-IRES-Cre;Camk2a-tTA;Ai93 mice and GCaMP6s-expressing Emx1-Cre;Camk2a-tTA;Ai94 mice. Several other combinations were absent of aberrant neural activity - including Emx1-CreERT2;Camk2a-tTA;Ai93 maintained on doxycycline, Camk2a-tTA;Ai93 with other Cre-drivers (Rorb-IRES2-Cre or Scnn1a-Tg3-Cre), Camk2-tTA with another GCaMP6s transgene (Camk2a-tTA;TRE-GCaMP6s line G6s2), Emx1-driven GCaMP6f (Emx1-Cre;Ai95) or GCaMP6s (Emx1-Cre;Ai96), excitatory neuron-expressing GCaMP6s strains (Snap25-2A-GCaMP6s and Thy1-GCaMP6s line GP4.3) and Pvalb-driven channelrhodopsin2 (Pvalb-Cre;Ai32).

Note on genetic background effect on tTA-induced neurotoxicity: Han et al. 2012 J Neurosci. 32:10574 reports mice expressing Tg(CaMK2a-tTA)1Mmay on the C57BL/6 background exhibit resistance to tTA-induced neurotoxicity (dentate gyrus granule cell layer atrophy). All other backgrounds tested (FVB/NJ, CBA/J, 129X1/SvJ, C3H/HeJ and DBA/1J) exhibit varying levels of neurotoxicity.

Note on Ai93D allele design: Prior to recombination, Ai93D mice have the TIGRE-Ins-TRE-LSL-GCaMP6f conditional allele, designed with a modified Tet response element (TRE or tetO) and loxP-flanked STOP cassette (LSL) upstream of GCaMP6f. Ai93D genotyping/sequencing revealed that two tandem copies of the TRE::floxed-STOP::GCaMP6f replacement vector were inserted during gene targeting. Therefore, the Ai93D allele is structurally composed of TIGRE-frt3-Ins-TRE-LSL-GCaMP6f-WPRE-bGHpA-Ins-AttB-PGK-5'hygro-SD-frt5-frt3-Ins-TRE-LSL-GCaMP6f-WPRE-bGHpA-Ins-AttL. The donating investigator reports that crossing Ai93D mice (Stock No. 024103) to several different Cre mouse lines consistently produces offspring with the expected GCaMP6f expression. Sufficiently strong and prolonged expression of Cre (as in the case of Cre-expressing mice such as CaMK2a-tTA) is likely to recombine between the furthest loxP sites; resulting in one copy of Tet-inducible TRE::GCaMP6f. While the floxed-STOP regions may individually recombine with weaker and/or transient Cre expression, the donating investigator suggests the Tet-dependent calcium activity monitoring function should not be adversely affected in those recombination scenarios (i.e., two copies of Tet-inducible TRE::GCaMP6f, or one copy of Tet-inducible TRE::GCaMP6f with the other copy retaining the floxed-STOP region).

Note on GCaMP6f: 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. 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 (with several amino acid substitutions designed to increase dynamic range and baseline fluorescence), and a rat calmodulin sequence modified to increase the fluorescence change for small calcium transients and accelerated kinetics. Other modifications in the cpEGFP-to-CaM linker also improve sensor function. 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.

As described for Stock No. 007004, the CaMK2a-tTA transgene integrated into chromosome 12 causing a 508.12 Kb deletion that spans the 3' half of Vipr2 (vasoactive intestinal peptide receptor 2), the entire Wdr60 (WD repeat domain 60), Esyt2 (extended synaptotagmin-like protein 2), D430020J02Rik (RIKEN cDNA D430020J02 gene) and Ncapg2 (non-SMC condensin II complex, subunit G2) loci and the first two exons of Ptprn2 (protein tyrosine phosphatase, receptor type, N polypeptide 2). Homozygous mice will therefore have a functional knock-out of the deleted loci (Wdr60, Esyt2, D430020J02Rik, Ncapg2), and altered or null expression of Vipr2 and Ptprn2. Founder line 1 has a copy number of greater than 20 [Goodwin et al. 2019 Genome Res. 29:494].

Development

The Ai93(TITL-GCaMP6f)-D;CaMK2a-tTA (or Ai93D;CaMK2a-tTA) double mutant animals were created in 2014 by breeding B6J.CaMK2a-tTA mice (Stock No. 007004) with B6;129.Ai93D mice (Stock No. 024103). The CaMK2a-tTA transgene and Ai93D allele are described below.

The CaMK2a-tTA transgene (CaMKIIα-tTA; Tg(CaMK2a-tTA)1Mmay) was designed by Dr. Mark Mayford (The Scripps Research Institute) to have (from 5' to 3') the ~8.5 kbp mouse CaMKIIalpha promoter, an artificial intron and splice sites, the tetracycline-regulated transactivator (tTA or "Tet-Off") gene and SV40 polyadenylation signal. Founder line B animals on a C57BL/6J genetic background (B6J.CaMK2a-tTA; Stock No. 007004) were used for creating the Ai93D;CaMK2a-tTA double mutant animals. As described for Stock No. 007004, the CaMK2a-tTA transgene integrated into chromosome 12 causing a 508.12 Kb deletion that spans the 3' half of Vipr2 (vasoactive intestinal peptide receptor 2), the entire Wdr60 (WD repeat domain 60), Esyt2 (extended synaptotagmin-like protein 2), D430020J02Rik (RIKEN cDNA D430020J02 gene) and Ncapg2 (non-SMC condensin II complex, subunit G2) loci and the first two exons of Ptprn2 (protein tyrosine phosphatase, receptor type, N polypeptide 2). Homozygous mice will therefore have a functional knock-out of the deleted loci (Wdr60, Esyt2, D430020J02Rik, Ncapg2), and altered or null expression of Vipr2 and Ptprn2. Founder line 1 has a copy number of greater than 20 [Goodwin et al. 2019 Genome Res. 29:494].

The Ai93(TITL-GCaMP6f)-D allele (Ai93D; Igs7^{tm93.1(tetO-GCaMP6f)Hze}) was created by Dr. Zeng and has two tandem copies of TRE::floxed-STOP::GCaMP6f 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). Therefore, the Ai93D allele is structurally composed of TIGRE-frt3-Ins-TRE-LSL-GCaMP6f-WPRE-bGHpA-Ins-AttB-PGK-5’hygro-SD-frt5-frt3-Ins-TRE-LSL-GCaMP6f-WPRE-bGHpA-Ins-AttL. The specific details of Ai93D are described for Stock No. 024103. B6;129.Ai93D mice from Stock No. 024103 were used for creating the Ai93D;R26-ZtTA double mutant animals.

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. 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.

The TRE promoter used here is Tet-responsive P_tight; containing a modified Tet response element (TRE_mod) composed of seven direct 36 bp repeats (each containing the 19 bp tet operator sequence [tetO]). TRE_mod is just upstream of a minimal human cytomegalovirus promoter (P_minCMVΔ), which lacks the enhancer that is part of the complete CMV promoter. Consequently, P_tight is silent in the absence of TetR or rTetR binding to tetO.

Allele

Symbol: Tg(Camk2a-tTA)1Mmay
Name: transgene insertion 1, Mark Mayford
MGI accession ID: MGI:2179066
Generation method: Transgenic
Attributes: Null/Knockout; Transactivator
Marker symbol: Tg(Camk2a-tTA)1Mmay
Marker name: transgene insertion 1, Mark Mayford
Chromosome: 12
Strain of origin: Not Specified
Expressed genes: tTA,tetracycline-controlled transactivator,E. coli
Site of expression: Expresses tTA in forebrain neurons.
Molecular note: The transgene contains the tetracycline-controlled transactivator protein (tTA) under regulatory control of the forebrain specific calcium/calmodulin-dependent kinase II promoter. The transgene integrated into chromosome 12 causing a 508.12 Kb deletion and disrupting the following genes: Vipr2, Wdr60, Esyt2, Ncapg2, and Ptprn2. The deletion results in a functional knock-out of Vipr2, Wdr60, Esyt2, and Ncapg2 in homozygous mice. Founder line 1 has a copy number of greater than 20.
General note: This transgene is line B.

Transgenic mice are viable, fertile, and display no overt phenotypic defects. Administration of tetracycline analogs such as doxycycline blocks transgene expression.

Allele

Symbol: Igs7^{tm93.1(tetO-GCaMP6f)Hze}
Name: targeted mutation 93, Hongkui Zeng
MGI accession ID: MGI:5558086
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Inducible
Marker symbol: Igs7
Marker name: intergenic site 7
Chromosome: 9
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: GCaMP,Genetically encoded calcium indicator,
Site of expression: Expression of fast variant calcium indicator (GCaMP6f) is under the control of tetO in cells/tissues where promoters driving Cre recombinase are expressed.
Molecular note: The targeting vector was designed with (from 5' to 3') an FRT3 site, two tandem copies of the chicken beta-globin HS4 insulator element (to reduce reporter gene expression in absence of transactivator protein), a modified Tet response element (TRE or tetO), a loxP-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), the GCaMP6 fast variant calcium indicator (GCaMP6f) coding sequence, a WPRE sequence (to enhance the mRNA transcript stability), a bovine growth hormone polyA signal, two tandem copies of the chicken beta-globin HS4 insulator element, an AttB site, a PGK-5' hygro cassette, an RNA splice donor and an FRT5 site 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. GCaMP6f has a chicken smooth muscle M13 fragment of myosin light chain kinase, a circularly permutated EGFP, and a rat calmodulin DNA fragment mutation for accelerated kinetics. Embryonic stem cells, previously targeted with FRT5-AttB-Hygro2 cassette-AttP-SV40pA-splice acceptor-PGKpA-FRT3 in the same locus, were re-transfected with the targeting vector and Flp recombinase vector. Genotyping/sequencing revealed that Ai93 had two tandem copies of the pTRE-LSL-GCaMP6f replacement vector inserted during RCME. Chimeric mice were bred with PhiC31 Gt(ROSA)26Sor^{tm3(phiC31*)Sor} mice to remove the AttB/AttP-flanked region. The Ai93D allele is therefore TIGRE-frt3-Ins-TRE-LSL-GCaMP6f-WPRE-bGHpA-Ins-AttB-PGK-5'hygro-SD-frt5-frt3-Ins-TRE-LSL-GCaMP6f-WPRE-bGHpA-Ins-AttL.