Overview

Also Known As:Ai94(TITL-GCaMP6s)-D;ROSA26-ZtTA

Ai94(TITL-GCaMP6s)-D;Rosa26-ZtTA (or Ai94D;Rosa26-ZtTA) 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 Cre-dependent tetracycline-controlled transactivator protein (tTA) inserted into the Gt(ROSA)26Sor locus on chromosome 6. 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.

Donating Investigator

Hongkui Zeng, Allen Institute for Brain Science

Genetic overview

Genetic Background	Generation

Gt(ROSA)26Sor^{tm5(ACTB-tTA)Luo}

Allele Type	Gene Symbol	Gene Name
Targeted (Reporter, Transactivator)	Gt(ROSA)26Sor	gene trap ROSA 26, Philippe Soriano

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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Research Applications

Research Tools
Neurobiology Research
Cancer Research

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$2,854.50 Domestic price Cryo Recovery

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Details

Detailed Description

Ai94(TITL-GCaMP6s)-D;Rosa26-ZtTA (or Ai94D;R26-ZtTA) 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 Cre-dependent tetracycline-controlled transactivator protein (tTA) inserted into the Gt(ROSA)26Sor locus on chromosome 6.

In the absence of Cre recombinase, Ai94D;R26-ZtTA mice are designed to have widespread lacZ expression but the expression of both GCaMP6s (EGFP fluorescence) and tTA are blocked by a floxed STOP cassette in each allele.

Following exposure of Ai94D;R26-ZtTA mice to Cre recombinase, the floxed-STOP cassette in each allele is deleted; leading to expression of tTA and GCaMP6s in the cre-expressing cells (see additional details below). GCaMP6s expression can then be regulated with tetracycline or its analog doxycycline (dox).

When Ai94D;R26-ZtTA 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;R26-ZtTA mice heterozygous for both targeted mutations are expected to be viable and fertile with no reported gross physical or behavioral abnormalities. The Ai94D donating investigator reports that mice homozygous for Ai94D are viable and fertile with no reported gross physical or behavioral abnormalities. The R26-ZtTA donating investigator reports that mice heterozygous for R26-ZtTA are more healthy and fertile than mice homozygous for R26-ZtTA.

For characterization information of Ai94 with other tTA and Cre lines, 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 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 GCaMP6s, 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.

Development

The Ai94(TITL-GCaMP6s)-D;Rosa26-ZtTA (or Ai94D;Rosa26-ZtTA) double mutant animals were created in 2015 by breeding mice harboring the R26-ZtTA allele (see Stock No. 012266) with B6J.Ai94D mice (Stock No. 024104). The R26-ZtTA allele and Ai94D allele are described below.

The Rosa26-ZtTA targeted mutation (R26-ZtTA; Gt(ROSA)26Sor^{tm5(ACTB-tTA)Luo}) was designed by Dr. Liqun Luo (Stanford University, HHMI) to have a CMV enhancer/chicken beta-actin core promoter (pCA), a loxP-flanked β-geo (lacZ/neo) transcriptional STOP cassette, the tetracycline-controlled transactivator protein (tTA) open reading frame, and an SV40 T-antigen poly(A) signal all inserted into Gt(ROSA)26Sor locus on chromosome 6. Mice harboring the ROSA26-ZtTA allele (on a mixed CD1, 129, C57BL/6 and/or FVB genetic background; see Stock No. 012266) were used for creating the Ai94D;R26-ZtTA double mutant animals.

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;R26-ZtTA 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	mice carrying this allele may be useful to generate compound mutant mice in which expression of a tetracycline-responsive promoter element (TRE; also called tet-operator or tetO)-driven gene of interest can be both directed to the cell types defined by the chosen Cre recombinase expression, as well as turned off by the addition of tetracycline (or its analog doxycycline [dox]).
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

Wild-type from the colony

Additional Information

Considerations for Choosing Controls

Genetics

Gt(ROSA)26Sor^{tm5(ACTB-tTA)Luo}

Allele Symbol: Gt(ROSA)26Sor^{tm5(ACTB-tTA)Luo}

Allele Name	targeted mutation 5, Liqun Luo
Allele Type	Targeted (Reporter, Transactivator)
Allele Synonym(s)	ROSA26-ZtTA; ZtTA
Gene Symbol and Name	Gt(ROSA)26Sor, gene trap ROSA 26, Philippe Soriano
Gene Synonym(s)
Promoter	ACTB, actin, beta, chicken
Expressed Gene	tTA, tetracycline-controlled transactivator, E. coli
Site of Expression	mice carrying this allele may be useful to generate compound mutant mice in which expression of a tetracycline-responsive promoter element (TRE; also called tet-operator or tetO)-driven gene of interest can be both directed to the cell types defined by the chosen Cre recombinase expression, as well as turned off by the addition of tetracycline (or its analog doxycycline [dox]).
Strain of Origin	(129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Chromosome	6
Molecular Note	The pCA-ZtTA construct was designed with a CMV enhancer/chicken beta-actin core promoter (pCA), a loxP-flanked beta-geo (lacZ-neomycin phosphotransferase fusion) transcriptional STOP cassette, the tetracycline-controlled transactivator protein (tTA or "Tet-Off") open reading frame, and an SV40 T-antigen poly(A) signal. This pCA-ZtTA construct was subcloned into the pROSA26-PA targeting vector to generate the final targeting construct pROSA26-ZtTA.
Mutations Made By	Liqun Luo, Stanford University

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

Mammalian Phenotype Terms by Genotype

References

Additional - Gt(ROSA)26Sor^{tm5(ACTB-tTA)Luo} related

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

Technical Support

CONTACT TECHNICAL SUPPORT

Genotyping Protocols
- Probe:GCaMP6s-Probe
- Standard PCR:Igs7-Alternate 1
- Standard PCR:Igs7-Alternate 1
- Probe:Gt(Rosa)26Sor(CAG) alternate 2
- Genotyping resources and troubleshooting
Breeding Considerations

When maintaining a live colony, mice heterozygous for both the Ai94D targeted mutation (Igs7^{tm94.1(tetO-GCaMP6s)Hze}) and also for the R26-ZtTA targeted mutation (Gt(ROSA)26Sor^{tm5(ACTB-tTA)Luo}) may be bred to wildtype mice from the colony or to C57BL/6J inbred mice. Alternatively, mice heterozygous or homozygous for Ai94D and heterozygous for R26-ZtTA may be bred to wildtype mice from the colony or to C57BL/6J inbred mice.
The Ai94D donating investigator reports that mice homozygous for Ai94D are viable and fertile with no reported gross physical or behavioral abnormalities. The R26-ZtTA donating investigator reports that mice heterozygous for R26-ZtTA are more healthy and fertile than mice homozygous for R26-ZtTA.
- Additional Breeding and Husbandry Support
Citation
When using the Ai94(TITL-GCaMP6s)-D;ROSA26-ZtTA mouse strain in a publication, please cite the originating article(s) and include JAX stock #024112 in your Materials and Methods section.

Animal Health Reports

Facility Barrier Level Descriptions

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

Pricing & Availability

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Service/Product	Description	Price
	Heterozygous for Gt(ROSA)26Sor<tm5(ACTB-tTA)Luo> and Igs7<tm94.1(tetO-GCaMP6s)Hze> or Heterozygous for Gt(ROSA)26Sor<tm5(ACTB-tTA)Luo> and wildtype for Igs7<tm94.1(tetO-GCaMP6s)Hze> or Wildtype for Gt(ROSA)26Sor<tm5(ACTB-tTA)Luo> and Heterozygous for Igs7<tm94.1(tetO-GCaMP6s)Hze> or Wildtype for Gt(ROSA)26Sor<tm5(ACTB-tTA)Luo> and Igs7<tm94.1(tetO-GCaMP6s)Hze>

Related Products and Services

Frozen Mouse Embryo	STOCK Gt(ROSA)26Sor<tm5(ACTB-tTA)Luo> Igs7<tm94.1(tetO-GCaMP	$2595.00
Frozen Mouse Embryo	STOCK Gt(ROSA)26Sor<tm5(ACTB-tTA)Luo> Igs7<tm94.1(tetO-GCaMP	$2595.00
Frozen Mouse Embryo	STOCK Gt(ROSA)26Sor<tm5(ACTB-tTA)Luo> Igs7<tm94.1(tetO-GCaMP	$3373.50
Frozen Mouse Embryo	STOCK Gt(ROSA)26Sor<tm5(ACTB-tTA)Luo> Igs7<tm94.1(tetO-GCaMP	$3373.50

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Also Known As:Ai94(TITL-GCaMP6s)-D;ROSA26-ZtTA

Donating Investigator

Genetic overview

Research Applications

Base Price

Detailed Description

Development

Expression Data

Control Suggestions

Additional Information

Gt(ROSA)26Sortm5(ACTB-tTA)Luo

Allele Symbol: Gt(ROSA)26Sortm5(ACTB-tTA)Luo

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 - Gt(ROSA)26Sortm5(ACTB-tTA)Luo related

Additional - Igs7tm94.1(tetO-GCaMP6s)Hze 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

Related Products and Services

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

Gt(ROSA)26Sor^{tm5(ACTB-tTA)Luo}

Allele Symbol: Gt(ROSA)26Sor^{tm5(ACTB-tTA)Luo}

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

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

Additional - Gt(ROSA)26Sor^{tm5(ACTB-tTA)Luo} related

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