B6.Cg-Snap25^tm1.1Hze/J

Stock number: 021879
Product name: Snap25-LSL-2A-EGFP-D
Research areas: Neurobiology Research, Research Tools

Description

The Snap25-LSL-2A-EGFP-D (also called Snap25-LSL-2A-EGFP-Δ or Snap25-LSL-2A-EGFP-Δneo/hygro) conditional allele is designed with a loxP-flanked STOP cassette and enhanced green fluorescent protein (EGFP) downstream of the synaptosomal-associated protein 25 (Snap25) translational STOP codon. Following Cre recombinase removal of the floxed STOP cassette, EGFP fluorescence is expected in cells/tissues where the expression patterns of Snap25 and Cre recombinase overlap. Snap25 is a t-SNARE protein. These mice may be useful as a Cre-lox tool for fluorescent visualization of synaptic vesicle/plasma membrane fusion.

Development

These Snap25-LSL-2A-EGFP-D mice (also called Snap25-LSL-2A-EGFP-Δ or Snap25-LSL-2A-EGFP-Δneo/hygro) have a loxP-flanked STOP cassette upstream of an enhanced greed fluorescent protein (EGFP), all inserted immediately downstream of the translational STOP codon of the synaptosomal-associated protein 25 gene (Snap25). The specific details are below.

The targeting vector contained, 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 to (but not including) the endogenous stop codon, a loxP-flanked STOP cassette that is in-frame with the upstream Snap25 exon 8 sequence (contains translational stop codons in all three reading frames and the PGK polyA sequence), an in-frame F2A peptide cleavage signal, an in frame EGFP gene, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a bovine growth hormone polyA signal, an AttB site, a PGK promoter-Neomycin resistance gene-PGK polyA cassette, an frt5 site, an mRNA splice acceptor, the 3' portion of the hygromycin gene with SV40 late polyA signal, and an AttP site.

This construct was electroporated into (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. Chimeric mice were bred to PhiC31-expressing mice (C57BL/6J congenic background; see Stock No. 007743) to remove the AttB/AttP-flanked sequences (PGK-Neo-polyA::frt5::RNA splice acceptor::3'hygro-polyA) and replace it with the recombined AttB/AttP site (AttL).

The resulting Snap25-LSL-2A-EGFP-D mice were bred with C57BL/6J wildtype mice for four more generations (and the PhiC31 transgene was removed) prior to sending to The Jackson Laboratory Repository in 2013. Upon arrival, males were used to cryopreserve sperm. To establish the living Snap25-LSL-2A-EGFP-D mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).

Allele

Symbol: Snap25^tm1.1Hze
Name: targeted mutation 1.1, Hongkui Zeng
MGI accession ID: MGI:5484607
Generation method: Targeted
Attributes: Reporter; Null/Knockout
Synonyms: Ai52; Ai52D; Snap25^{tm1.1(xstpx-EGFP)Hze}; Snap25-2A; Snap25-LSL-2A-EGFP; Snap25-LSL-2A-EGFP-Delta; Snap25-LSL-2A-EGFP-DeltaNeo/Hygro
Marker symbol: Snap25
Marker name: synaptosomal-associated protein 25
Marker synonyms: bA416N4.2; Bdr; CMS18; dJ1068F16.2; GENA 70; RIC4; RIC-4; SEC9; SNAP; SNAP-25; SNAP-25a; SNAP-25B; sp; SUP
Chromosome: 2
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: GFP,Green Fluorescent Protein,
Site of expression: EGFP fluorescence is expected in diverse neuronal subpopulations.
Molecular note: The targeting vector contained, 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 to (but not including) the endogenous stop codon, a loxP-flanked STOP cassette that is in-frame with the upstream Snap25 exon 8 sequence (contains translational stop codons in all three reading frames and the PGK polyA sequence), an in-frame F2A peptide cleavage signal, an in frame EGFP gene, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a bovine growth hormone polyA signal, an AttB site, a PGK promoter-Neomycin resistance gene-PGK polyA cassette, an frt5 site, an mRNA splice acceptor, the 3' portion of the hygromycin gene with SV40 late polyA signal, and an AttP site. PhiC31-mediated recombination removed the AttB/AttP-flanked sequences (PGK-Neo-polyA::frt5::RNA splice acceptor::3'hygro-polyA) and replaced it with the recombined AttB/AttP site (AttL).