B6(SJL)-Gabra2^tm2.1Uru/J

Stock number: 037213
Product name: α2^fl
Research areas: Neurobiology Research, Research Tools

Description

The α2^fl mice have loxP sites flanking exon 5 of the Gabra2 gene. These mice may be useful for studying physiologic and therapeutic properties of GABA_A α-subunits in such diseases as epilepsy or substance abuse disorders.

Detailed description

The neurotransmitter, γ-aminobutyric acid (GABA), acts as the primary inhibitory neurotransmitter in the central nervous system. GABA binds to either GABA_A or GABA_B receptors, resulting in an intracellular environment that inhibits neuronal signaling. There are 5 subunits that make up a GABA_A receptor, frequently two α-, two β-, and one γ-subunit. These subunits form a transmembrane pore that regulates flow of chloride ions. Each subunit is coded by a different gene and multiple variants of each subunit exist. The α-subunits are integral for the receptor as they are involved with both binding GABA (αβ-interface) and other chemicals such as benzodiazepines (αγ-interface). There are 6 known variants of α subunits where four have been determined to be benzodiazepine-sensitive (α1, α2, α3 and α5) and two (α4 and α6) to be non-responsive to benzodiazepines such as Diazepam. The Gabra2 gene encodes the α2 subunit specifically.

α2^fl mice possess loxP sites flanking exon 5 of the Gabra2 gene. The donating investigator reports that when these α2^fl mice were bred to EIIa-Cre transgenic mice (JAX Stock No: 003724) to produce a Gabra2 global knock-out; no detectable levels of α2 mRNA were found in cortex, spinal cord or dorsal root ganglion (DRG) tissues. α2^fl mice are viable and fertile with no overt phenotype. When bred to mice that express tissue-specific Cre recombinase, resulting offspring will have exon 5 deleted in the cre-expressing tissues.

GABA_A receptors are expressed throughout the spinal cord dorsal horn, specifically the α2 GABA_A receptors and play a role in regulation of nociceptive signals. Positive allosteric modulation of GABA_A receptors with benzodiazepines have been used to reverse heightened pain responses in models of inflammatory and neuropathic pain. To further investigate the role of α2 GABA_A receptors in spinal cord pain, the donating investigator crossed the α2^fl mice to sensory neuron-specific sodium channel (sns)-Cre transgenic mice. The resulting sns-α2 ^-/- mice demonstrated a significant reduction in α2-subunit mRNA expression in DRG as compared to wildtype controls. The sns-α2 ^-/- mice displayed responses to noxious thermal (heat), mechanical and chemical stimuli similar to wildtype controls. However, after intrathecal injections of Diazepam, the sns-α2 ^-/- mice showed a reduced response to the analgesic effects of Diazepam compared to controls.

Conditional knockouts of the α2 subunit in principal neurons of the dentate gyrus and the hippocampal CA3 region resulted in reduced sensitivity to the anxiolytic-like action of Diazepam, whereas a conditional knockout of the α2 subunit in the hippocampal CA1 regions resulted in a reduced sensitivity to the fear-reducing-like action of Diazepam.

These α2^fl mice may be useful for studying therapeutic targets for schizophrenia, epilepsy, substance abuse disorders, anxiety and fear-related disorders or other processes regulated by the GABAergic system.

The donating investigator has made several GABA_A α-subunit mouse lines available: α1(H101R) KI (Stock No. 037209), α2(H101R)KI (Stock No. 037210), α3(H126R) KI (Stock No. 037211), α5(H105R) KI (Stock No. 037212), α2^fl (Stock No. α3 KO (Stock No. 037213), and 037214) and Gabra5^fl (Stock No. 031135).

Development

The α2^fl targeting vector was designed to insert a loxP site upstream of exon 5 and a FRT-flanked neomycin selection cassette, followed by a second loxP site, downstream of exon 5 of the Gabra2 gene. The construct was electroporated into C57BL/6N-derived embryonic stem (ES) cells (Eurogentec). Correctly targeted ES cells were injected into recipient blastocysts and the resulting chimeric mice were bred to ACTFlpe mice (Stock No. 005703) to remove the neo-cassette. The ACTFlpe transgene was removed through selective breeding to C57BL/6J (Stock No. 000664). The resulting colony of α2^fl mice, with neo cassette removed and the floxed exon 5 intact, were maintained on the C57BL/6J background by the donating laboratory (see SNP note below). Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes.

Of note, the Y chromosome was fixed during back-crossing to C57BL/6J.



In 2022, a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. One of the 43 markers throughout the genome was segregating with SJL, on Chromosome 12.

Allele

Symbol: Gabra2^tm2.1Uru
Name: targeted mutation 2.1, Uwe Rudolph
MGI accession ID: MGI:5140553
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Synonyms: alpha2^fl
Marker symbol: Gabra2
Marker name: gamma-aminobutyric acid (GABA) A receptor, subunit alpha 2
Marker synonyms: C630048P16Rik; DEE78; EIEE78; Gabra-2
Chromosome: 5
Strain of origin: C57BL/6N
Molecular note: A loxP site was inserted upstream of exon 5. An FRT-flanked neo cassette with a 5' loxP site was inserted downstream of exon 5. Flp-mediated recombination removed the neo cassette and left exon 5 floxed.