B6;129S-Slc17a6^{tm1.1(flpo)Hze}/J

Stock number: 030212
Product name: Slc17a6-IRES2-FlpO-D (Vglut2-IRES2-FLPo-D)
Research areas: Neurobiology Research, Research Tools

Description

Slc17a6-IRES2-FlpO-D knock-in mice are designed to have optimized FLP recombinase expression directed to VGLUT2-expressing cells. FlpO expression/activity largely recapitulates the endogenous Slc17a6 gene in the brain - widespread throughout most of the brain, except very sparse expression in striatum and restricted populations within cerebellum, medulla and pons. The specific cell types identified to have FLP expression are excitatory glutamatergic, VGLUT2+ neuron cell bodies throughout the brain. These mice may be used to generate conditional mutations for studying gain-or-loss of function and/or fate mapping related to glutamatergic neurons.

Detailed description

The vesicular glutamate transporter 2 (VGLUT2; encoded by Slc17a6) mediates the uptake of glutamate into synaptic vesicles at presynaptic nerve terminals of excitatory neural cells.

The Slc17a6-IRES2-FlpO-D knock-in mutation has an IRES2 sequence and an optimized FLP recombinase (FlpO) gene inserted downstream of the Slc17a6 translational STOP codon - this is designed to have the endogenous promoter/enhancer elements direct FlpO expression to VGLUT2+ cells. When Slc17a6-IRES2-FlpO-D mice are bred with mice containing frt-flanked sequences, FlpO-mediated recombination will result in deletion of the frt-flanked sequences in Slc17a6-expressing cells in the offspring.

The donating investigator reports FlpO recombinase expression/activity largely recapitulates the endogenous Slc17a6 gene - widespread throughout most of the brain, except very sparse expression in striatum and restricted populations within cerebellum, medulla and pons. The specific cell types identified to have FLP expression are excitatory glutamatergic, VGLUT2+ neuron cell bodies throughout the brain. The donating investigator did not examine FlpO expression in tissues other than brain. This knock-in allele is designed to avoid disrupting expression of the endogenous protein; although Slc17a6 expression from the Slc17a6-IRES2-FlpO-D allele has not been evaluated to date (March 2017). Heterozygous mice are viable and fertile with no reported gross physical or behavioral abnormalities. The Jackson Laboratory colony tested breeding homozygous mice and found them to be viable and fertile.

For characterization information, see images at the Allen Institute for Brain Science website (Slc17a6-IRES2-FlpO-D images).

Development

The Slc17a6-IRES2-FlpO-D mice (also called Vglut2-IRES2-FlpO-D, Slc17a6-IRES2-FlpO-Δ or Slc17a6-IRES2-FlpO-Δneo) were created in the laboratory of Dr. Hongkui Zeng (Allen Institute for Brain Science) to have an IRES2 sequence and an optimized FLP recombinase (flpO) gene inserted immediately downstream of the translational STOP codon of the solute carrier family 17 (sodium-dependent inorganic phosphate cotransporter) member 6 gene(Slc17a6; Vglut2) on chromosome 7. The specific details are below.

The targeting vector contained, from 5' to 3', a partial Slc17a6 sequence spanning exon 12 (including the endogenous stop codon), an internal ribosome entry site 2 sequence (IRES2; allows translation initiation in the middle of an mRNA sequence), a FlpO recombinase gene (a codon-optimized FLPe gene modified for translation in mammalian cells and higher recombination efficiency in cells and in mice), 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 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-pA::frt5::RNA splice acceptor::3'hygro-pA) and replace it with the recombined AttB/AttP site (AttL). The resulting Slc17a6-IRES2-FlpO-D mice were bred with C57BL/6J wildtype mice for several generations (and the PhiC31 transgene was removed). In 2017, 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 Slc17a6-IRES2-FlpO-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: Slc17a6^{tm1.1(flpo)Hze}
Name: targeted mutation 1.1, Hongkui Zeng
MGI accession ID: MGI:5903982
Generation method: Targeted
Attributes: Recombinase-expressing
Synonyms: Slc17a6-IRES2-FlpO-D; Vglut2-IRES2-FLPo-D
Marker symbol: Slc17a6
Marker name: solute carrier family 17 (sodium-dependent inorganic phosphate cotransporter), member 6
Marker synonyms: 2900073D12Rik; Dnpi; VGLUT2
Chromosome: 7
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: FLP,FLP recombinase,Saccharomyces cerevisiae
Site of expression: When these mice are bred with mice containing frt-flanked sequences, FlpO-mediated recombination will result in deletion of the frt-flanked sequences in Slc17a6-expressing cells in the offspring. FlpO recombinase expression/activity is widespread throughout most of the brain
Molecular note: The targeting vector contains, from 5' to 3', a partial Slc17a6 sequence spanning exon 12 (including the endogenous stop codon), an internal ribosome entry site 2 sequence, a FlpO recombinase gene (a codon-optimized FLPe gene modified for translation in mammalian cells and higher recombination efficiency in cells and in mice), 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 polyA signal, and an AttP site. PhiC31-mediated recombination removed the AttB/AttP-flanked sequence (PGK-Neo-polyA::frt5::RNA splice acceptor::3'hygro-polyA) and replaced it with the recombined AttB/AttP site (AttL).