D2.129S6(B6)-Slc17a6^tm2(cre)Lowl/MwarUgfmJ

Stock number: 038999
Product name: DBA/2J VGlut2-IRES-Cre
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

Vglut2-ires-Cre knock-in mice have Cre recombinase expression directed to excitatory glutamatergic neuron cell bodies, without disrupting endogenous vesicular glutamate transporter 2 expression. These mice may be used to generate conditional mutations for studying gain-or-loss of function and/or fate mapping related to glutamatergic neurons. The DBA/2J background of this line (Stock No. 038999) has made it particularly useful in studies of glaucoma.
See Development section 2025 information regarding its partial neo/kan sequence.

The Jackson Laboratory Repository has the Vglut2-ires-Cre knock-in allele Slc17a6^tm2(cre)Lowl from Dr. Bradford Lowell on a C57BL/6;FVB;129S6 genetic background (Stock No. 016963), on a C57BL/6J congenic background (Stock No. 028863) and on a DBA/2J congenic background (Stock No. 038999).

Detailed description

DBA/2J-VGlut2-Cre congenic mice were generated through repeated crosses of Vglut2-ires-cre knock-in (C57BL/6J) (Stock No. 028863) animals to inbred DBA/2J mice (Stock No. 000671). The DBA/2J background of this line has made it particularly useful in studies of glaucoma. When crossed to DBA/2J-OMM-GFP mice (038998), resultant progeny can be used to identify retinal ganglion cell (RGC) soma and axons prior to and during glaucomatous degeneration. They can also be used to purify specifically RGC mitochondria from retina or optic nerve via an immuno pull down (PMID 31501572; Fecher, 2019). Heterozygotes are viable, fertile, and without overt abnormalities. Homozygous viability/fertility has not been assessed, but are anticipated to be normal (November, 2023).

The original Vglut2-ires-Cr3e allele expresses Cre recombinase in excitatory glutamatergic neuron cell bodies without disrupting endogenous vesicular glutamate transporter 2 expression. Specifically, Cre recombinase activity is detected in excitatory glutamatergic, VGLUT2 positive, neuron cell bodies in the thalamus, paraventricular nucleus, nucleus of the lateral olfactory tract, basolateral nucleus of the amygdala, ventromedial hypothalamus, piriform cortex, posterior hypothalamus, ventral premammillary nucleus, subthalamic nucleus, medial geniculate nucleus, reticulotegmental nucleus, pontine gray, external cuneate nucleus, and lateral reticular nucleus. When crossed with a strain containing loxP sequences, Cre-mediated recombination results in tissue-specific deletion of flanked sequences in the offspring. In the DBA/2J background line, only RGCs have been examined for tissue-specific deletion. The iris pigment deposition, increase in intraocular pressure (IOP) and RGC degeneration have not been analyzed in this strain but are expected to match the parent DBA/2J strain. Homozygous Vglut2-ires-Cre knock-in mice are viable and fertile with no reported abnormalities.

In an attempt to offer alleles on well-characterized or multiple genetic backgrounds, alleles are frequently moved to a genetic background different from that on which an allele was first characterized. This is the case for the strain above. It should be noted that the phenotype could vary from that originally described. We will modify the strain description, if necessary, as published results become available.

Development

DBA/2J-VGlut2-Cre congenic mice were generated through crosses of Vglut2-ires-cre knock-in (C57BL/6J) (Stock No. 028863) animals to inbred DBA/2J mice (Stock No. 000671) for 11 generations in the laboratory of Dr. Romain Cartoni (then at Duke University). Upon arrival at The Jackson Laboratory, sperm was cryopreserved. Cryorecovery/live colony development will involve DBA/2J (Stock No. 000671).

The original Vglut2-ires-Cre knock-in allele was designed by Dr. Bradford Lowell (Beth Israel Deaconess Medical Center / Harvard). A targeting vector was designed to insert an internal ribosome entry site-linked Cre recombinase gene (and frt-flanked kan/neo - see 2024 and 2025 information below) downstream of the stop codon of the solute carrier family 17 (sodium-dependent inorganic phosphate cotransporter), member 6 gene (Slc17a6 or VGLUT2) on chromosome 7. The construct was electroporated into 129S6/SvEvTac derived W4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. The resulting chimeric animals were crossed to C57BL/6 mice, and then crossed to FVB. In 2012, Vglut2-ires-Cre knock-in mice on a mixed C57BL/6;FVB;129S6 genetic background (with black coat color) were sent to The Jackson Laboratory Repository. Upon arrival, sperm was cryopreserved. An aliquot of that frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664) and rederive our live colony as Stock No. 016963, which was thereafter maintained by breeding mutant mice together.

In 2015, Dr. Melissa R. Warden (Cornell University) purchased some Stock No. 016963 animals (total backcross generation N1 onto C57BL/6J). They further backcrossed them to C57BL/6J inbred mice for five additional generations. The resulting C57BL/6J-congenic strain, called Vglut2-ires-Cre knock-in (C57BL/6J), were sent to The Jackson Laboratory Repository in 2016 as Stock No. 028863.

In 2024, the Vglut2-ires-Cre allele was found to have a kan/neo sequence. Additional details are below.
In 2024, expanded quality control screening at The Jackson Laboratory revealed that Stock No. 038999 DBA/2J VGlut2-IRES-Cre heterozygotes genotyped positive for a kanamycin/neomycin resistance (kan/neo) sequence, while their wildtype littermates genotyped negative for kan/neo. Testing of the two other VGlut2-IRES-Cre strains (Stock Nos. 016963/028863) also showed as kan/neo positive. Subsequent communication with the donating investigator confirmed the targeting vector contained IRES-Cre followed by a frt-flanked kan/neo cassette.

In 2025, the Vglut2-ires-Cre allele was found to have a single frt site followed by a partial kan/neo sequence. Additional details are below.
In 2025, The Jackson Laboratory bred some Stock No. 028863 C57BL/6J VGlut2-IRES-Cre to a germline FLP in an attempt to generate a new strain with the kan/neo cassette removed. After several generations, the new strain's offspring continued to test positive for our Generic Neo [probe] assay. Subsequently, we sequenced some Stock No. 028863 homozygous mice and found the allele contained (from 5' to 3') IRES, SV40 NLS, 1030 bp Cre variant with flanking 259 bp sequence, HSVTKpA, 346 bp multiple cloning site remnant, SV40pA, frt, 221 bp partial neo/kan. No additional frt sites were detected. This suggests the original targeting vector may have contained a frt-flanked full-length neo/kan that the donating lab removed via germline FLP recombinase exposure, but in doing so, a 221 bp neo/kan remnant inserted downstream of the one remaining frt site. It has not been determined if presence of the 221 bp partial neo/kan has any adverse effect on expression of the VGlut2-IRES-Cre allele.

Allele

Symbol: Slc17a6^tm2(cre)Lowl
Name: targeted mutation 2, Bradford B Lowell
MGI accession ID: MGI:5141269
Generation method: Targeted
Attributes: Recombinase-expressing
Synonyms: Vglut2^Cre; Vglut2-ires-Cre
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
Expressed genes: cre,cre recombinase,bacteriophage P1
Site of expression: Cre recombinase activity is detected in excitatory glutamatergic, VGLUT2 positive, neuron cell bodies in the thalamus, paraventricular nucleus, nucleus of the lateral olfactory tract, basolateral nucleus of the amygdala, ventromedial hypothalamus, piriform cortex, posterior hypothalamus, ventral premammillary nucleus, subthalamic nucleus, medial geniculate nucleus, reticulotegmental nucleus, pontine gray, external cuneate nucleus, and lateral reticular nucleus.
Molecular note: A targeting vector, constructed from a BAC clone containing the Slc17a6 (Vglut2) gene, was utilized to insert an IRES-SV40 NLS-Cre-polyA STOPs-frt-221bp partial kan/neo fragment into the Slc17a6 locus just downstream of the endogenous stop codon. Cre is expressed from the Slc17a6 gene promoter.