Overview

Also Known As: Snap25-2A-GCaMP6s-D

Snap25-2A-GCaMP6s-D knockin mice are a calcium-indicator tool strain. These mice are designed to have widespread brain expression of the GCaMP6 slow variant calcium indicator (GCaMP6s; an ultrasensitive detector of single neuronal action potentials with slower decay and response kinetics) that results in EGFP expression following calcium binding (such as neuronal activation). These mice may also be useful for studying t-SNARE proteins and synaptic vesicle/plasma membrane fusion.

Donating Investigator

Hongkui Zeng, Allen Institute for Brain Science

Genetic overview

Genetic Background	Generation
	N6F1+pN4 (2019-01-15 00:00:00)

Snap25^tm3.1Hze

Allele Type	Gene Symbol	Gene Name
Targeted (Reporter)	Snap25	synaptosomal-associated protein 25

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Research Applications

Research Tools
Neurobiology Research
Cell Biology Research

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Base Price

Starting at:

$278.00 Domestic price for female

356.51 Domestic price for breeder pair

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Details

Detailed Description

The Snap25-2A-GCaMP6s-D knockin allele has a viral 2A oligopeptide sequence (T2A) that mediates ribosomal skipping and a GCaMP6 slow variant calcium indicator (GCaMP6s), all inserted downstream of the synaptosomal-associated protein 25 (Snap25) coding region. This is designed to have widespread brain expression of the GCaMP6 slow variant calcium indicator (GCaMP6s; an ultrasensitive detector of single neuronal action potentials with slower decay and response kinetics) that results in EGFP expression following calcium binding (such as neuronal activation).

Functional testing/characterization of Snap25-2A-GCaMP6s-D mice has not yet been completed (October 2014). While they are expected to have pan-neuronal GCaMP6s expression, native fluorescence and immunohistochemical staining (via anti-GFP antibody) are not yet defined. Like other GCaMP6s-expressing mice from the same donating investigator, Snap25-2A-GCaMP6s-D cells can be expected to have low EGFP expression throughout the brain in the absence of calcium binding. Following calcium binding (such as neuronal activation), significantly increased EGFP expression is expected. Heterozygous mice are viable and fertile with no reported gross physical or behavioral abnormalities. To date (August 2017), it has not been attempted to generate homozygous mice by the donating investigator or The Jackson Laboratory.

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 Snap25-2A-GCaMP6s-D targeted mutation (also called Snap25-2A-GCaMP6s-Δhygro) has a viral 2A oligopeptide sequence (T2A) that mediates ribosomal skipping and a GCaMP6 slow variant calcium indicator (GCaMP6s), all inserted downstream of the synaptosomal-associated protein 25 (Snap25) coding region. The specific details are below.

(129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells, already targeted with a LSL-2A-EGFP vector downstream of the Snap25 coding region (see Stock No. 021879), were re-targeted with a "T2A-GCaMP6s" vector and a FLP-expressing plasmid to facilitate recombination.

Correctly re-targeted ES cells had (from 5' to 3')
a partial Snap25 sequence spanning intron 6 through intron 7,
an frt3 site within Snap25 intron 7,
a partial Snap25 exon 8 sequence up (but not including) the endogenous translational stop codon,
a T2A peptide cleavage signal that is in-frame with the Snap25 coding sequence,
a GCaMP6s gene (described below) that is in-frame with the Snap25 coding sequence,
a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability),
a bovine growth hormone polyA sequence,
an AttB site,
a PGK promoter-hygromycin resistance gene-SV40polyA cassette (with an mRNA splice donor-frt5 site-mRNA splice acceptor in the hygromycin gene),
and an AttP site.

Correctly re-targeted ES cells were injected into recipient blastocysts. The resulting chimeric animals were bred to PhiC31-expressing mice (C57BL/6J congenic background; see Stock No. 007743) to delete the AttB/AttP-flanked sequences (PGK-5'hygro::mRNA splice donor::frt5::mRNA splice acceptor::3'hygro::SV40 polyA) and replace it with the recombined AttB/AttP site (AttL).

The resulting Snap25-2A-GCaMP6s-D mice were bred with C57BL/6J wildtype mice for several generations (and the PhiC31 gene was removed) prior to sending generation N6F1 males to The Jackson Laboratory Repository in 2015. Upon arrival, males were used to cryopreserve sperm. To establish the living Snap25-2A-GCaMP6s-D mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).

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	EGFP expression is expected following calcium binding (such as neuronal activation) in the brain.

Control Suggestions

Wild-type from the colony

Approximate Controls

000664 C57BL/6J

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

Snap25^tm3.1Hze

Allele Symbol: Snap25^tm3.1Hze

Allele Name	targeted mutation 3.1, Hongkui Zeng
Allele Type	Targeted (Reporter)
Allele Synonym(s)	Snap25^tm3.1Hze; targeted mutation 3.1, Hongkui Zeng
Gene Symbol and Name	Snap25, synaptosomal-associated protein 25
Gene Synonym(s)	Bdr; blind drunk; SNAP; bA416N4.2; SNAP-25; dJ1068F16.2; RIC-4; RIC4; SEC9; sp; GENA 70; Bdr; SNAP-25B; SNAP-25a; CMS18; SUP
Expressed Gene	GFP, Green Fluorescent Protein,
Site of Expression	EGFP expression is expected following calcium binding (such as neuronal activation) in the brain.
Strain of Origin	(129S6/SvEvTac x C57BL/6NCrl)F1
Chromosome	2
Molecular Note	The targeting construct contained (from 5' to 3') a partial sequence spanning intron 6 through intron 7, an frt3 site within intron 7, a partial exon 8 sequence up to but not including the endogenous translational stop codon, a T2A peptide cleavage signal in-frame with the coding sequence, a GCaMP6s gene (slow variant calcium indicator; an ultrasensitive detector of single neuronal action potentials with slow response kinetics) that is in-frame with the coding sequence, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a bovine growth hormone polyA sequence, an AttB site, a PGK promoter-hygromycin resistance gene-SV40polyA cassette (with an mRNA splice donor-frt5 site-mRNA splice acceptor in the hygromycin gene), and an AttP site. PhiC31-mediated recombination removed the selection cassette.

Disease/Phenotype

Disease Terms

Research Areas By Genotype

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

Research Tools
- Fluorescent Proteins
- Cell Biology Research
- Genetics Research
  - Tissue/Cell Markers: neurons
  - Tissue/Cell Markers
- Neurobiology Research
  - cell marker
Neurobiology Research
- Fluorescent protein expression in neural tissue
- Optogenetic and Chemogenetic tools
  - Calcium sensor expressing strains
- Channel and Transporter Defects
  - calcium
- Neurotransmitter Receptor and Synaptic Vesicle Defects
Cell Biology Research
- Channel and Transporter Defects
  - calcium
- Vesicular Trafficking

Mammalian Phenotype Terms by Genotype

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

Genotype: Snap25^tm3.1Hze/Snap25⁺
B6.Cg-Snap25/J

mammalian phenotype

no phenotypic analysis

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 - Snap25^tm3.1Hze related

Technical Support

Contact Technical Support

Genotyping Protocols
- Standard PCR: Snap25^{tm3.1(GCaMP6s)Hze}-Alternate 1
- Genotyping resources and troubleshooting
Dietary Information
- New Diet as of March 2015: Lab Diet® 5K0Q (6% fat)
Breeding Considerations

When maintaining a live colony, heterozygous mice may be bred to wildtype siblings or to C57BL/6J inbred mice (Stock No. 000664). To date (August 2017), it has not been attempted to generate homozygous mice by the donating investigator or The Jackson Laboratory.
- Additional Breeding and Husbandry Support
Mating System
- Wild-type x Heterozygote
- Heterozygote x Wild-type
Citation
When using the Snap25-2A-GCaMP6s-D mouse strain in a publication, please cite the originating article(s) and include JAX stock #025111 in your Materials and Methods section.

Animal Health Reports

Facility Barrier Level Descriptions

AX18 (Maximum)

Pricing & Availability

Available

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Live Mouse

Age

Genotype

Price

weeks

Breeder Pair

Sex

Genotype

Price

Female
Male

Cryorecovery - Not-For-Profit & Academic Pricing

Service	Genotype	Price
	Heterozygous or wildtype for Snap25<tm3.1Hze>

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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Frozen Mouse Embryo

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Also Known As: Snap25-2A-GCaMP6s-D

Donating Investigator

Genetic overview

Research Applications

Base Price

Detailed Description

Development

Expression Data

Control Suggestions

Approximate Controls

Additional Information

Selected References

Snap25tm3.1Hze

Allele Symbol: Snap25tm3.1Hze

Disease Terms

Research Areas By Genotype

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

Mammalian Phenotype Terms by Genotype

Genotype: Snap25tm3.1Hze/Snap25+B6.Cg-Snap25/J

mammalian phenotype

References

Additional - Snap25tm3.1Hze related

Genotyping Protocols

Dietary Information

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

Snap25^tm3.1Hze

Allele Symbol: Snap25^tm3.1Hze

Genotype: Snap25^tm3.1Hze/Snap25⁺
B6.Cg-Snap25/J

Additional - Snap25^tm3.1Hze related