B6J.129S6(FVB)-Slc32a1^tm2(cre)Lowl/MwarJ

Stock number: 028862
Product name: Vgat-ires-cre knock-in (C57BL/6J)
Research areas: Cancer Research, Neurobiology Research, Research Tools

Description

Vgat-ires-Cre knock-in mice have Cre recombinase expression directed to inhibitory GABAergic neuron cell bodies, without disrupting endogenous vesicular inhibitory amino acid transporter expression. These mice may be used to generate conditional mutations for studying gain-or-loss of function and/or fate mapping related to GABAergic neuronal function.

Detailed description

Vgat-ires-Cre knock-in mice have Cre recombinase expression directed to inhibitory GABAergic neuron cell bodies, without disrupting endogenous vesicular inhibitory amino acid transporter expression. Both heterozygous and homozygous mice are viable and fertile with no gross physical abnormalities. Specifically, Cre recombinase activity is detected in inhibitory GABAergic neuron cell bodies in the caudate putamen, suprachiasmatic nucleus, central amygdaloid nucleus, and zona incerta, nucleus accumbens, lateral septum, medial septum, reticular nucleus of the thalamus, substantia nigra pars reticulata, and Purkinje cell layer of the cerebellum. When crossed with a strain containing loxP site-flanked sequences, Cre-mediated recombination results in tissue-specific deletion of flanked sequences in the offspring.

Based on crosses with Ai14 tdTomato reporter mice, the donating investigator of this C57BL/6J-congenic line (Stock No. 028862 Vgat-ires-cre knock-in (C57BL/6J)) reports that these mice have the same cre expression compared to the parental line (Stock No. 016962 C57BL/6;FVB;129S6 Vgat-ires-Cre knock-in).

Also of note, the targeted Slc32a1 gene is located ~3.5 kbp (~1.9 cM) from the nonagouti locus (A). Whereas the parental Stock No. 016962 has agouti coat color (presumably due to the presence of 129S6-derived A^w allele from the ES cells used in creating the Vgat-ires-Cre allele), the donating investigator of this C57BL/6J-congenic Stock No. 028862 reports that during backcrossing they successfully got C57BL/6J recombination encompassing the a locus and bred to homozygosity. Therefore, Stock No. 028862 is a/a (black coat color).

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. The phenotype described above is for Vgat-ires-Cre knock-in mice on a mixed C57BL/6;FVB;129S6 genetic background (Stock No. 016962). It should be noted that the phenotype of this C57BL/6J-congenic line (Stock No. 028862 Vgat-ires-cre knock-in (C57BL/6J)) could vary from that originally described for mice on other genetic backgrounds. We will modify the strain description if necessary as published results become available.

Development

The Vgat-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 downstream of the stop codon of the solute carrier family 32 (GABA vesicular transporter), member 1 gene (Slc32a1 or VGAT) on chromosome 2. 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, Vgat-ires-Cre knock-in mice on a mixed C57BL/6;FVB;129S6 genetic background 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. 016962, which was thereafter maintained by breeding mutant mice together.


In 2013, Dr. Melissa R. Warden (Cornell University) purchased some Stock No. 016962 animals (total backcross generation N1 onto C57BL/6J). They further backcrossed them to C57BL/6J inbred mice for at least nine additional generations. The resulting C57BL/6J-congenic strain, called Vgat-ires-cre knock-in (C57BL/6J), were sent to The Jackson Laboratory Repository in 2016 as Stock No. 028862. Upon arrival, males were used to cryopreserve sperm. To establish the living mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred mice (Stock No. 000664).


Of note, Dr. Warden's lab 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).
In 2016, a 144 SNP analysis (with markers covering all 19 chromosomes and the X chromosome) was performed on the first generation of rederived mice at The Jackson Laboratory Repository. This showed the genetic background was 99.3-99.65% C57BL/6J allele-type markers.

Allele

Symbol: Slc32a1^tm2(cre)Lowl
Name: targeted mutation 2, Bradford B Lowell
MGI accession ID: MGI:5141270
Generation method: Targeted
Attributes: Recombinase-expressing
Marker symbol: Slc32a1
Marker name: solute carrier family 32 (GABA vesicular transporter), member 1
Chromosome: 2
Strain of origin: 129S6/SvEvTac
Expressed genes: cre,cre recombinase,bacteriophage P1
Site of expression: Cre recombinase activity is detected in inhibitory GABAergic neuron cell bodies in the caudate putamen, suprachiasmatic nucleus, central amygdaloid nucleus, and zona incerta, nucleus accumbens, lateral septum, medial septum, reticular nucleus of the thalamus, substantia nigra pars reticulata, and Purkinje cell layer of the cerebellum.
Molecular note: A targeting vector, constructed from a BAC clone containing the Slc32a1 (Vgat) gene, was utilized to insert an internal ribosomal entry site followed by the cre recombinase sequence into the Slc32a1 locus just downstream of the endogenous stop codon. Cre is expressed from the Slc32a1 gene promoter. Immunohistochemistry on brain and Western blot analysis shows decreased expression of SLC32A1 protein in homozygotes.