Overview

Also Known As: B6;129S-Gt(ROSA)26Sor^{tm96.1(CAG-GCaMP6s)Hze}/J, Ai96(RCL-GCaMP6s) or Ai96

This Ai96 allele is available on a C57BL/6J genetic background as Stock No. 028866 and that version will replace this mixed background strain in the future.

Ai96(RCL-GCaMP6s) (also called Ai96) mice are a Cre-dependent, fluorescent, calcium-indicator tool strain. Ai96 has a floxed-STOP cassette preventing transcription of the GCaMP6 slow variant calcium indicator (GCaMP6s; an ultrasensitive detector of single neuronal action potentials with slower decay and response kinetics). After Cre exposure, increased EGFP fluorescence is observed following calcium binding (such as neuronal activation).

Donating Investigator

Hongkui Zeng, Allen Institute for Brain Science

Genetic overview

Genetic Background	Generation
	N1+pN4F8 (2019-06-12 00:00:00)

Gt(ROSA)26Sor^{tm96(CAG-GCaMP6s)Hze}

Allele Type	Gene Symbol	Gene Name
Targeted (Conditional ready (e.g. floxed), Reporter, Inserted expressed sequence)	Gt(ROSA)26Sor	gene trap ROSA 26, Philippe Soriano

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Research Applications

Research Tools
Neurobiology Research

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Base Price

Starting at:

$333.00 Domestic price for female 4-week

411.51 Domestic price for breeder pair

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Details

Detailed Description

Ai96(RCL-GCaMP6s) mice (also called Ai96) harbor the Rosa-CAG-LSL-GCaMP6s conditional allele, designed with a floxed-STOP cassette upstream of the GCaMP6 slow variant calcium indicator (GCaMP6s; see detailed description below).
Although under control of the endogenous Gt(ROSA)26Sor promoter/enhancer regions and the CAG hybrid promoter, widespread expression of GCaMP6s is prevented by STOP cassettes. After exposure to Cre recombinase, EGFP fluorescence is observed following calcium binding (such as neuronal activation).

Specifically, the donating investigator reports Ai96 mice have no reported levels of EGFP fluorescence prior to exposure to Cre recombinase. When Ai96 mice are bred with a Cre-driver line to create double transgenic animals (GCaMP6s+/Cre+), cells expressing Cre exhibit low EGFP fluorescence in the absence of calcium binding. Following calcium binding (such as neuronal activation), increased EGFP fluorescence is observed in these cells.

The donating investigator also reports that following neuronal activation, the fluorescent intensity observed in double transgenic Ai96 mice (GCaMP6s+/Cre+) is less than that of the double transgenic Ai95 system using GCaMP6 fast variant calcium indicator (GCaMP6f+/Cre+ ; see Stock No. 024105). Although both systems utilize Cre-dependent GCaMP6/EGFP fusion genes, they differ in the kinetics and sensitivity of their GCaMP6 variants.

Heterozygous Ai96 mice are viable and fertile with no reported gross physical or behavioral abnormalities. The donating investigator has not attempted to generate homozygous Ai96 mice to date (March 2014).

For characterization information, see images at the Allen Institute for Brain Science website (Ai96(RCL-GCaMP6s) images).

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.

Development

The Ai96(RCL-GCaMP6s) allele (also called Ai96) was designed by Dr. Hongkui Zeng (Allen Institute for Brain Science) to have a floxed-STOP cassette upstream of the GCaMP6 slow variant calcium indicator (GCaMP6s), all inserted into the Gt(ROSA)26Sor locus. Specific details below.

The Rosa-pCAG-LSL-GCaMP6s-WPRE-bGHpA-AttB-PGK-neo-polyA-AttP targeting vector was designed with (from 5' to 3')
a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS),
a loxP-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), the GCaMP6s coding sequence, a WPRE sequence (to enhance mRNA transcript stability), a bovine growth hormone polyA signal,
the GCaMP6s coding sequence (described below),
a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance mRNA transcript stability),
a bovine growth hormone polyA signal,
an AttB site,
a PGK promoter-Neomycin resistance gene-PGK polyA cassette,
and an AttP site.
This entire targeting vector was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus via electroporation of (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. Correctly targeted ES cells were selected. The resulting Ai96 mice were bred to C57BL/6J wildtype mice for at least one additional generation.
In 2014, Ai96 heterozygous males on a mixed genetic background (estimated 75% C57BL/6 ; with black coat color) were sent to The Jackson Laboratory Repository. Upon arrival, sperm was cryopreserved. An aliquot of that frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664) and rederive our live colony, which was thereafter maintained by breeding heterozygous mice with wildtype mice from the colony or with C57BL/6J inbred mice.

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.

Expression Data

Expressed Gene	GFP, Green Fluorescent Protein,
Site of Expression	After exposure to Cre recombinase, EGFP fluorescence is observed following calcium binding (such as neuronal activation).

Control Suggestions

Wild-type from the colony

Additional Information

Considerations for Choosing Controls

Selected References

Madisen L; Garner AR; Shimaoka D; Chuong AS; Klapoetke NC; Li L; van der Bourg A; Niino Y; Egolf L; Monetti C; Gu H; Mills M; Cheng A; Tasic B; Nguyen TN; Sunkin SM; Benucci A; Nagy A; Miyawaki A; Helmchen F; Empson RM; Knopfel T; Boyden ES; Reid RC; Carandini M; Zeng H. 2015. Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance. Neuron 85(5):942-58PubMed: 25741722 MGI: J:219930

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Genetics

Gt(ROSA)26Sor^{tm96(CAG-GCaMP6s)Hze}

Allele Symbol: Gt(ROSA)26Sor^{tm96(CAG-GCaMP6s)Hze}

Allele Name	targeted mutation 96, Hongkui Zeng
Allele Type	Targeted (Conditional ready (e.g. floxed), Reporter, Inserted expressed sequence)
Allele Synonym(s)	Gt(ROSA)26Sor^{tm96(CAG-GCaMP6s)Hze}; targeted mutation 96, Hongkui Zeng
Gene Symbol and Name	Gt(ROSA)26Sor, gene trap ROSA 26, Philippe Soriano
Gene Synonym(s)	beta geo; ROSA26; R26; Gtrgeo26; Gtrosa26; Gtrgeo26; Gtrosa26; AV258896; expressed sequence AV258896; gene trap ROSA 26; gene trap ROSA b-geo 26; SETD5-AS1; Thumpd3as1
Promoter	CAG, CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter,
Expressed Gene	GFP, Green Fluorescent Protein,
Site of Expression	After exposure to Cre recombinase, EGFP fluorescence is observed following calcium binding (such as neuronal activation).
Strain of Origin	(129S6/SvEvTac x C57BL/6NCrl)F1
Chromosome	6
Molecular Note	The targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), a loxP-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), the GCaMP6s coding sequence, a WPRE sequence (to enhance mRNA transcript stability), a bovine growth hormone polyA signal, the GCaMP6s coding sequence (described below), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance mRNA transcript stability), a bovine growth hormone polyA signal, an AttB site, a PGK promoter-Neomycin resistance gene-PGK polyA cassette, and an AttP site. This entire targeting vector was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus. GCaMP6s has a chicken smooth muscle M13 fragment of myosin light chain kinase, a circularly permutated EGFP, and a rat calmodulin DNA fragment to increase the fluorescence change for small calcium transients.

Disease/Phenotype

Disease Terms

Research Areas By Genotype

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

Research Tools
- Diabetes and Obesity Research
  - loxP
- Genetics Research
  - Mutagenesis and Transgenesis
  - Tissue/Cell Markers: cell marker for bone marrow transplantation
  - Tissue/Cell Markers: transplantation marker for embryonic and adult tissue
  - Mutagenesis and Transgenesis: Cre-lox System
  - Tissue/Cell Markers
  - Tissue/Cell Markers: Cre-lox System
- Cre-lox System
  - loxP-flanked Sequences
  - loxP-flanked Sequences: Test/Reporter
- Developmental Biology Research
  - transplantation marker for embryonic and adult tissue
  - Cre-lox System
- Reproductive Biology Research
  - transplantation marker for embryonic and adult tissue
  - Cre-lox System
- Fluorescent Proteins
Neurobiology Research
- Cre-lox System
  - loxP-flanked Sequences
  - loxP-flanked Sequences: Test/Reporter
- Optogenetic and Chemogenetic tools
  - Cre-dependent optogenetic tools
  - Calcium sensor expressing strains
- Fluorescent protein expression in neural tissue

Mammalian Phenotype Terms by Genotype

The following phenotype relates to a compound genotype created using this strain

Genotype: Gt(ROSA)26Sor^{tm96(CAG-GCaMP6s)Hze}/Gt(ROSA)26Sor⁺
involves: 129S6/SvEvTac * C57BL/6

normal phenotype

no abnormal phenotype detected
- mice are viable and fertile

References

Madisen L; Garner AR; Shimaoka D; Chuong AS; Klapoetke NC; Li L; van der Bourg A; Niino Y; Egolf L; Monetti C; Gu H; Mills M; Cheng A; Tasic B; Nguyen TN; Sunkin SM; Benucci A; Nagy A; Miyawaki A; Helmchen F; Empson RM; Knopfel T; Boyden ES; Reid RC; Carandini M; Zeng H. 2015. Transgenic mice for intersectional targeting of neural sensors and effectors with high specificity and performance. Neuron 85(5):942-58PubMed: 25741722 MGI: J:219930

Additional References

Chen TW; Wardill TJ; Sun Y; Pulver SR; Renninger SL; Baohan A; Schreiter ER; Kerr RA; Orger MB; Jayaraman V; Looger LL; Svoboda K; Kim DS. 2013. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499(7458):295-300PubMed: 23868258 MGI: J:203036

Additional - Gt(ROSA)26Sor^{tm96(CAG-GCaMP6s)Hze} related

Technical Support

Contact Technical Support

Genotyping Protocols
- SEPARATED MELT: Gt(ROSA)26Sor^{tm95.1(CAG-GCaMP6f)Hze}-alternate 2
- Genotyping resources and troubleshooting
Breeding Considerations

When maintaining a live colony, heterozygous mice may be bred to wildtype mice from the colony or to C57BL/6J inbred mice (Stock No. 000664). The donating investigator has not attempted to generate homozygous mice to date (March 2014).
- Additional Breeding and Husbandry Support
Mating System
- Wild-type x Heterozygote
- Heterozygote x Wild-type
Citation
When using the Ai96(RCL-GCaMP6s) or Ai96 mouse strain in a publication, please cite the originating article(s) and include JAX stock #024106 in your Materials and Methods section.

Animal Health Reports

Facility Barrier Level Descriptions

AX11 (Maximum)

Pricing & Availability

Available

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Genotype

Price

weeks

Breeder Pair

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Genotype

Price

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Cryorecovery - Not-For-Profit & Academic Pricing

Service	Genotype	Price
	Heterozygous or Wildtype for Gt(ROSA)26Sor<tm96(CAG-GCaMP6s)Hze>

We will fulfill your order by providing at least two carriers for each strain ordered. The total number, sex, and genotypes provided will vary, although typically 8 or more animals are provided. Please check genotypes which will be recovered. While the genotypes of all animals produced will be communicated to you prior to scheduling shipment, the genotypes of animals provided may not reflect the mating scheme and genotypes described in the strain description. Animals are typically ready to ship in 11-14 weeks. If a second recovery is required to produce the minimum number of animals, then delivery time would increase to approximately 25 weeks. If we fail to produce animals of the correct genotype, you will not be charged. We cannot guarantee the reproductive success of mice shipped to your facility. If the mice are lost after the first three days (post-arrival) or do not produce progeny at your facility, a new order and fee will be necessary.

Cryorecovery to establish a Dedicated Supply for greater quantities of mice. Mice recovered can be used to establish a dedicated colony to contractually supply you mice according to your requirements. Price by quotation.

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Also Known As: B6;129S-Gt(ROSA)26Sortm96.1(CAG-GCaMP6s)Hze/J, Ai96(RCL-GCaMP6s) or Ai96

Donating Investigator

Genetic overview

Research Applications

Base Price

Detailed Description

Development

Expression Data

Control Suggestions

Additional Information

Selected References

Gt(ROSA)26Sortm96(CAG-GCaMP6s)Hze

Allele Symbol: Gt(ROSA)26Sortm96(CAG-GCaMP6s)Hze

Disease Terms

Research Areas By Genotype

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

Mammalian Phenotype Terms by Genotype

Genotype: Gt(ROSA)26Sortm96(CAG-GCaMP6s)Hze/Gt(ROSA)26Sor+involves: 129S6/SvEvTac * C57BL/6

normal phenotype

References

Additional References

Additional - Gt(ROSA)26Sortm96(CAG-GCaMP6s)Hze related

Genotyping Protocols

Breeding Considerations

Mating System

Citation

Animal Health Reports

Live Mouse

Breeder Pair

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

Also Known As: B6;129S-Gt(ROSA)26Sor^{tm96.1(CAG-GCaMP6s)Hze}/J, Ai96(RCL-GCaMP6s) or Ai96

Gt(ROSA)26Sor^{tm96(CAG-GCaMP6s)Hze}

Allele Symbol: Gt(ROSA)26Sor^{tm96(CAG-GCaMP6s)Hze}

Genotype: Gt(ROSA)26Sor^{tm96(CAG-GCaMP6s)Hze}/Gt(ROSA)26Sor⁺
involves: 129S6/SvEvTac * C57BL/6

Additional - Gt(ROSA)26Sor^{tm96(CAG-GCaMP6s)Hze} related