Overview

Also Known As:Ai94(TITL-GCaMP6s)-D;CaMK2a-tTA

Ai94(TITL-GCaMP6s)-D;CaMK2a-tTA (or Ai94D;CaMK2a-tTA) mice have a Cre/Tet-dependent, fluorescent calcium indicator GCaMP6s 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 slow variant calcium indicator (GCaMP6s; an ultrasensitive detector of single neuronal action potentials with slower decay and response kinetics). Following subsequent calcium binding (such as neuronal activation), increased EGFP fluorescence is observed in those cells.

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].

Donating Investigator

Dr. Mark Mayford, The Scripps Research Institute

Hongkui Zeng, Allen Institute for Brain Science

Genetic overview

Genetic Background	Generation

Tg(Camk2a-tTA)1Mmay

Allele Type
Transgenic (Null/Knockout, Transactivator)

Igs7^{tm94.1(tetO-GCaMP6s)Hze}

Allele Type	Gene Symbol	Gene Name
Targeted (Conditional ready (e.g. floxed), Reporter, Inducible)	Igs7	intergenic site 7

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Details

Detailed Description

Ai94(TITL-GCaMP6s)-D;CaMK2a-tTA (or Ai94D;CaMK2a-tTA) mice have a Cre/Tet-dependent, fluorescent calcium indicator GCaMP6s (an ultrasensitive detector of single neuronal action potentials with slower decay and 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, Ai94D;CaMK2a-tTA mice are designed to have tTA expression in forebrain neurons but GCaMP6s expression (EGFP fluorescence) blocked by a floxed STOP cassette. Of note, the similarly designed Ai62(TITL-tdT)-D reporter (Stock No. 022731) combined with the same Camk2a-tTA transgene (Stock No. 007004) had offspring reported to have a few very weakly tdTomato-positive cells in cortex and hippocampus.
Following exposure of Ai94D;CaMK2a-tTA mice to Cre recombinase, the floxed STOP cassette in Ai94D is deleted; leading to expression of GCaMP6s in forebrain neurons. GCaMP6s expression can be regulated with tetracycline or its analog doxycycline (dox).

When Ai94D;CaMK2a-tTA mice are bred with Cre-driver lines to create triple transgenic animals (GCaMP6s+/tTA+/Cre+), EGFP fluorescence in cre-expressing cells is low in the absence of calcium binding. Following calcium binding (such as neuronal activation), increased EGFP fluorescence is observed in those cells. See further phenotype considerations below.

Ai94D;CaMK2a-tTA mice heterozygous or homozygous for Ai94D and hemizygous for CaMK2a-tTA are viable and fertile with no reported gross physical or behavioral abnormalities. To date (October 2016), it has not been attempted to make Ai94D;CaMK2a-tTA animals homozygous for the CaMK2a-tTA transgene.

For characterization information, see images at the Allen Institute for Brain Science website (Ai94(TITL-GCaMP6s) images).

Further phenotype considerations: Steinmetz et al. 2017 ENeuro reports aberrant neural activity is occasionally observed in Emx1-Cre;Camk2a-tTA;Ai94 mice. This paper is summarized below.
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 GCaMP6s: 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. The GCaMP6s fusion gene was designed by Drs. Douglas Kim and Loren Looger (Janelia Farm, HHMI). Compared to GCaMP6f, the slower kinetics render GCaMP6s more sensitive and slower to decay. GCaMP6s has a chicken smooth muscle M13 fragment of myosin light chain kinase, a circularly permutated EGFP (with several amino acid substitutions designed to optimize dynamic range, baseline fluorescence and sensitivity), and a rat calmodulin sequence modified to increase the fluorescence change for small calcium transients. 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 and disrupting the genes Vipr2 (vasoactive intestinal peptide receptor 2), Wdr60 (WD repeat domain 60), Esyt2 (extended synaptotagmin-like protein 2), Ncapg2 (non-SMC condensin II complex, subunit G2), and Ptprn2 (protein tyrosine phosphatase, receptor type, N polypeptide 2). 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 [Goodwin et al. 2019 Genome Res. 29:494].

Development

The Ai94(TITL-GCaMP6s)-D;CaMK2a-tTA (or Ai94D;CaMK2a-tTA) double mutant animals were created in 2015 by breeding B6J.CaMK2a-tTA mice (Stock No. 007004) with B6J.Ai94D mice (Stock No. 024104). The CaMK2a-tTA transgene and Ai94D 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 Ai94D;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 Ai94(TITL-GCaMP6s)-D allele (Ai94D; Igs7^{tm94.1(tetO-GCaMP6s)Hze}) was created by Dr. Hongkui Zeng (Allen Institute for Brain Science) and has TRE::floxed-STOP::GCaMP6s 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 Ai94D allele is structurally composed of TIGRE-frt3-Ins-TRE-LSL-GCaMP6s-WPRE-bGHpA-Ins-AttL. The specific details of Ai94D are described for Stock No. 024104. B6J.Ai94D mice from Stock No. 024104 were used for creating the Ai94D;CaMK2a-tTA double mutant animals.

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). 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.

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.

Expression Data

Expressed Gene	tTA, tetracycline-controlled transactivator, E. coli
Site of Expression	Expresses tTA in forebrain neurons.
Expressed Gene	GCaMP, Genetically encoded calcium indicator,
Site of Expression	Expression of fast variant calcium indicator (GCaMP6s) is under the control of tetO in cells/tissues where promoters driving Cre recombinase are expressed.

Control Suggestions

Noncarrier

Approximate Controls

000664 C57BL/6J

Additional Information

Considerations for Choosing Controls

Genetics

Tg(Camk2a-tTA)1Mmay

Allele Symbol: Tg(Camk2a-tTA)1Mmay

Allele Name	transgene insertion 1, Mark Mayford
Allele Type	Transgenic (Null/Knockout, Transactivator)
Allele Synonym(s)	CamDAT; CaMKIIalpha-tTA; CaMKII-tTA; CAMK-rTA; line B; pCaMKII-tTA; Tg^{(CaMKIItTA)Mmay}; Tg^{alpha-CaMkII-tTA}
Gene Symbol and Name	Tg(Camk2a-tTA)1Mmay, transgene insertion 1, Mark Mayford
Gene Synonym(s)
Promoter	Camk2a, calcium/calmodulin-dependent protein kinase II alpha, mouse, laboratory
Expressed Gene	tTA, tetracycline-controlled transactivator, E. coli
Site of Expression	Expresses tTA in forebrain neurons.
Strain of Origin	Not Specified
Chromosome	12
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.
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.
Mutations Made By	Dr. Mark Mayford, The Scripps Research Institute

Igs7^{tm94.1(tetO-GCaMP6s)Hze}

Allele Symbol: Igs7^{tm94.1(tetO-GCaMP6s)Hze}

Allele Name	targeted mutation 94.1, Hongkui Zeng
Allele Type	Targeted (Conditional ready (e.g. floxed), Reporter, Inducible)
Allele Synonym(s)	Ai94(TIGRE-Ins-TRE-LSL-GCaMP6s)-D; Ai94(TITL-GCaMP6s); Ai94D
Gene Symbol and Name	Igs7, intergenic site 7
Gene Synonym(s)
Promoter	tetO, tet operator,
Expressed Gene	GCaMP, Genetically encoded calcium indicator,
Site of Expression	Expression of fast variant calcium indicator (GCaMP6s) is under the control of tetO in cells/tissues where promoters driving Cre recombinase are expressed.
Strain of Origin	(129S6/SvEvTac x C57BL/6NCrl)F1
Chromosome	9
Molecular Note	The 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 modified Tet response element (TRE or tetO), a floxed-STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), the GCaMP6 slow variant calcium indicator coding sequence, a WPRE sequence (to enhance mRNA stability), a BGH polyA, two copies of chicken beta-globin HS4 insulator element, an AttB site, a PGK-5'hygro cassette, an RNA splice donor and a FRT5 site. The GCaMP6 slow variant calcium indicator (GCaMP6s) is an ultrasensitive detector of single neuronal action potentials with slow response kinetics and is an improved version of GCaMP5G. 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 pTRE-LSL-GCaMP6s replacement vector and Flp recombinase vector for recombinase-mediated cassette exchange (RCME).Chimeric mice were bred with PhiC31 Gt(ROSA)26Sor^{tm3(phiC31*)Sor} mice to remove the AttB/AttP-flanked PGK-hygromycin-SV40polyA cassette and replace it with the recombined AttB/AttP site (AttL).

Disease/Phenotype

Disease Terms

Research Areas By Phenotype

This mouse can be used to support research in many areas including:

Research Tools
- Tet Expression Systems
  - tTA/rtTA Responsive Strains
  - tTA/rtTA Expressing Strains
- Fluorescent Proteins
- Developmental Biology Research
  - Cre-lox System
  - transplantation marker for embryonic and adult tissue
- Genetics Research
  - Mutagenesis and Transgenesis: Cre-lox System
  - Tissue/Cell Markers: Cre-lox System
  - Tissue/Cell Markers
  - Tissue/Cell Markers: neurons
  - Tissue/Cell Markers: transplantation marker for embryonic and adult tissue
  - Mutagenesis and Transgenesis: Tetop Tet System
- Neurobiology Research
  - cell marker
  - Tetop Tet System
- Cancer Research
  - Cre-lox System
  - Tetop Tet System
- Cre-lox System
  - loxP-flanked Sequences
  - loxP-flanked Sequences: Test/Reporter
- Cardiovascular Research
  - Cre-lox System
  - Tetop Tet System
Neurobiology Research
- Tet Expression System
  - tTA/rtTA Expressing Strains
  - tTA/rtTA Responsive Strains
- Fluorescent protein expression in neural tissue
- Cre-lox System
  - loxP-flanked Sequences
  - loxP-flanked Sequences: Test/Reporter
- Optogenetic and Chemogenetic tools
  - Cre-dependent optogenetic tools
  - Calcium sensor expressing strains
Cancer Research
- Cre-lox System
  - loxP-flanked Sequences

Mammalian Phenotype Terms by Genotype

References

Additional - Igs7^{tm94.1(tetO-GCaMP6s)Hze} related

Additional - Tg(Camk2a-tTA)1Mmay related

Technical Support

CONTACT TECHNICAL SUPPORT

Genotyping Protocols
- Standard PCR:Tg(Camk2a-tTA)1Mmay
- Probe:GCaMP6s-Probe
- Standard PCR:Igs7-Alternate 1
- Genotyping resources and troubleshooting
Breeding Considerations

When maintaining a live colony, mice heterozygous or homozygous for the Ai94D targeted mutation (Igs7^{tm94.1(tetO-GCaMP6s)Hze}) and hemizygous for the CaMK2a-tTA transgene (Tg(Camk2a-tTA)1Mmay) may be bred to mice heterozygous or homozygous for Ai94D and wildtype (noncarrier) for CaMK2a-tTA.

Stock No. 024115: Ai94D;CaMK2a-tTA mice heterozygous or homozygous for Ai94D and hemizygous for CaMK2a-tTA are viable and fertile with no reported gross physical or behavioral abnormalities. To date (October 2016), it has not been attempted to make Ai94D;CaMK2a-tTA animals homozygous for the CaMK2a-tTA transgene.

Regarding viability and fertility of the single mutant parental mouse lines:

For the single mutant parental mouse line for Ai94D (Stock No. 024104), the donating investigator reports that mice homozygous for Ai94D are viable and fertile with no reported gross physical or behavioral abnormalities. Similarly in 2016, Stock No. 024104 at The Jackson Laboratory Repository is viable and fertile as homozygotes.

For the CaMK2a-tTA parental mouse line (Stock No. 007004), it has not been attempted to make the CaMK2a-tTA transgenic mouse line homozygous to date (October 2016).
- Additional Breeding and Husbandry Support
Citation
When using the Ai94(TITL-GCaMP6s)-D;CaMK2a-tTA mouse strain in a publication, please cite the originating article(s) and include JAX stock #024115 in your Materials and Methods section.

Animal Health Reports

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Production of mice from cryopreserved embryos or sperm occurs in a maximum barrier room, G200

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	Heterozygous for Igs7<tm94.1(tetO-GCaMP6s)Hze> and Hemizygous for Tg(Camk2a-tTA)1Mmay or Heterozygous for Igs7<tm94.1(tetO-GCaMP6s)Hze> and non carrier for Tg(Camk2a-tTA)1Mmay

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Also Known As:Ai94(TITL-GCaMP6s)-D;CaMK2a-tTA

Donating Investigator

Genetic overview

Research Applications

Base Price

Detailed Description

Development

Expression Data

Control Suggestions

Approximate Controls

Additional Information

Tg(Camk2a-tTA)1Mmay

Allele Symbol: Tg(Camk2a-tTA)1Mmay

Igs7tm94.1(tetO-GCaMP6s)Hze

Allele Symbol: Igs7tm94.1(tetO-GCaMP6s)Hze

Disease Terms

Research Areas By Phenotype

This mouse can be used to support research in many areas including:

Mammalian Phenotype Terms by Genotype

References

Additional - Igs7tm94.1(tetO-GCaMP6s)Hze related

Additional - Tg(Camk2a-tTA)1Mmay related

Genotyping Protocols

Breeding Considerations

Citation

Animal Health Reports

Production of mice from cryopreserved embryos or sperm occurs in a maximum barrier room, G200

Live Mouse

Cryorecovery - Not-For-Profit & Academic Pricing

Payment Terms and Conditions

The Jackson Laboratory's Genotype Promise

Terms Of Use

Additional Use Restrictions Apply

Licensing Information

JAX® Mice, Products & Services Conditions of Use

No Warranty

Credit for PRODUCTS or SERVICES

Animal Care and Use for SERVICES

No Liability

Igs7^{tm94.1(tetO-GCaMP6s)Hze}

Allele Symbol: Igs7^{tm94.1(tetO-GCaMP6s)Hze}

Additional - Igs7^{tm94.1(tetO-GCaMP6s)Hze} related