B6;129S-Slc17a8^{tm1.1(cre)Hze}/J

Stock number: 028534
Product name: Slc17a8-IRES2-Cre-D (or Vglut3-IRES2-Cre-D)
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

Slc17a8-IRES2-Cre-D knockin mice have Cre recombinase expression directed to Vglut3-expressing cells, without disrupting endogenous vesicular glutamate transporter 3 expression. These mice may be used to generate conditional mutations for studying gain-or-loss of function and/or fate mapping related to glutamatergic synaptic vesicle trafficking.

Detailed description

The Slc17a8 gene encodes the vesicular glutamate transporter 3 (Vglut3) protein that mediates synaptic transmission by transporting the neurotransmitter, glutamate, into secretory vesicles.

The Slc17a8-IRES2-Cre-D knockin mutation has an IRES2 sequence and a Cre recombinase gene inserted downstream of the translational STOP codon of the Slc17a8 gene. As such, Slc17a8-IRES2-Cre-D mice have both endogenous gene and Cre recombinase expression directed to Vglut3-expressing cells by the endogenous promoter/enhancer elements of the Slc17a8 locus. When Slc17a8-IRES2-Cre-D mice are bred with mice containing loxP-flanked sequences, Cre-mediated recombination will result in deletion of the floxed sequences in Vglut3-expressing cells in the offspring. The donating investigator reports Cre recombinase expression and activity largely recapitulates the endogenous Slc17a8 gene's adult expression pattern in areas such as cortex, cerebral nuclei, thalamus and midbrain. In addition, the Cre reporter expression pattern also appears to be somewhat broader than the endogenous pattern. The donating investigator did not examine cre expression in tissues other than brain. Heterozygous mice are viable and fertile with no reported gross physical or behavioral abnormalities. To date (December 2015), it has not been attempted to make this strain homozygous.

Development

These Slc17a8-IRES2-Cre-D mice (also called Slc17a8-IRES2-Cre-Δ or Slc17a8-IRES2-Cre-Δneo/hygro) have an IRES2 sequence and a Cre recombinase gene inserted immediately downstream of the translational STOP codon of the svesicular glutamate transporter 3 gene (Slc17a8 or Vglut3) on chromosome 10. The specific details are below.

The targeting vector contained, from 5' to 3', a partial Slc17a8 sequence spanning intron 11, an frt3 site within Slc17a8 intron 11, a partial Slc17a8 exon 12 sequence up to and including the endogenous stop codon, an internal ribosome entry site 2 (IRES2) sequence (allows translation initiation in the middle of an mRNA sequence), a Cre recombinase gene, a bovine growth hormone polyA sequence, an AttB site, a PGK promoter-Neomycin resistance gene-PGK polyA cassette, an frt5 site, an mRNA splice acceptor sequence, the 3' portion of the hygromycin gene (Hygro2) 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 Slc17a8-IRES2-Cre-D mice were bred with C57BL/6J wildtype mice for several generations (and the PhiC31 transgene was removed). In 2016, male mice from generation N3 with black coat color were sent to The Jackson Laboratory Repository. Upon arrival, males are used to cryopreserve sperm. To establish the living Slc17a8-IRES2-Cre-D mouse colony, an aliquot of the frozen sperm is used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).


Of note, the donating investigator reports that, at least once during backcrossing, a heterozygous female was bred to a C57BL/6J inbred male (thus the Y chromosome of the congenic strain is of C57BL/6J origin).

Allele

Symbol: Slc17a8^{tm1.1(cre)Hze}
Name: targeted mutation 1.1, Hongkui Zeng
MGI accession ID: MGI:5792821
Generation method: Targeted
Attributes: Recombinase-expressing; No functional change
Synonyms: Slc17a8-IRES2-Cre-D; Vglut3-IRES2-Cre-D
Marker symbol: Slc17a8
Marker name: solute carrier family 17 (sodium-dependent inorganic phosphate cotransporter), member 8
Marker synonyms: DFNA25; VGLUT3; Vgt3
Chromosome: 10
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: Slc17a8,solute carrier family 17 (sodium-dependent inorganic phosphate cotransporter), member 8,mouse, laboratory; cre,cre recombinase,bacteriophage P1
Site of expression: When these mice are bred with mice containing loxP-flanked sequences, Cre recombinase expression and activity largely recapitulates the endogenous Slc17a8 gene's adult expression pattern in areas such as cortex, cerebral nuclei, thalamus and midbrain.
Molecular note: The targeting vector containes, from 5' to 3', a partial Slc17a8 sequence spanning intron 11, an frt3 site within Slc17a8 intron 11, a partial Slc17a8 exon 12 sequence up to and including the endogenous stop codon, an IRES2 sequence, a Cre recombinase, a bovine growth hormone polyA sequence, an AttB site, a PGK promoter-Neomycin resistance gene-PGK polyA cassette, an frt5 site, an mRNA splice acceptor sequence, the 3' portion of the hygromycin gene (Hygro2) with SV40 late polyA signal, and an AttP site. Chimeric mice were bred to PhiC31-expressing mice 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). Mice have both endogenous gene and Cre recombinase expression directed to Vglut3-expressing cells by the endogenous promoter/enhancer elements of the Slc17a8 locus.