B6.Cg-Snap25^tm3.1Hze/J

Stock number: 025111
Product name: Snap25-2A-GCaMP6s-D
Research areas: Cell Biology Research, Neurobiology Research, Research Tools

Description

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.

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.

Allele

Symbol: Snap25^tm3.1Hze
Name: targeted mutation 3.1, Hongkui Zeng
MGI accession ID: MGI:5613588
Generation method: Targeted
Attributes: Reporter
Marker symbol: Snap25
Marker name: synaptosomal-associated protein 25
Chromosome: 2
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: GFP,Green Fluorescent Protein,
Site of expression: EGFP expression is expected following calcium binding (such as neuronal activation) in the brain.
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.