B6.129(Cg)-Gabra2^tm1.1Uru/J

Stock number: 037210
Product name: α2(H101R)
Research areas: Neurobiology Research

Description

α2(H101R) knock-in mice were generated by incorporating a point mutation in exon 4 of the Gabra2 gene, which encodes a single amino acid substitution (Histidine-101 to Arginine). 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.

This α2(H101R) knock-in model includes a point mutation in exon 4 of the Gabra2 gene that results in replacement of Histidine-101 with Arginine, rendering the α2 subunit to be like the benzodiazepine-insensitive subunits α4 and α6. The donating investigator reports that these knock-in mice, on a 129/SvJ background or on a C57BL/6J background, do not demonstrate an overt phenotype and response to GABA is indistinguishable between α2(H101R) knock-in mice and wildtype controls. In contrast to α1(H101R) (Stock No. 037209) and α3(H126R) (Stock No. 037211) knock-in lines, the α2(H101R) mice do not display anxiolytic behaviors after Diazepam exposure when performing light-dark choice test and elevated plus maze test. However, the α2(H101R) mice do show anxiolytic-like response after injection with sodium phenobarbital on the same behavioral assays, suggesting that the altered behavior is due to dysfunction at benzodiazepine-receptor interface. No significant changes in Diazepam-induced sedation, motor impairment or anticonvulsant response between α2(H101R) knock-in mice and wildtype controls. These mice may be useful for studying therapeutic targets for epilepsy or substance abuse disorders.

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. 037213), α3 KO (Stock No. 037214) and Gabra5^fl (Stock No. 031135).

Development

The α2(H101R) knock-in allele was created by introducing a point mutation (Histidine-101 to Arginine) into exon 4 of the Gabra2 gene and a loxP-flanked neomycin resistance marker into an engineered Sal I site in intron 3. The sequence TTT CAC AAT in exon 4 encoding the amino acids FHN (positions 100 to 102) was mutated to TTC CGG AAT encoding the amino acids FRN. The construct was electroporated into 129/OlaHsd-derived E14 embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6J blastocysts and the resulting chimeric mice were identified by genotyping and bred to EIIa-cre transgenic mice on a 129/Sv background. The cre transgene was removed through selective breeding to 129/Sv mice. As per donating investigator the 129/Sv mice utilized for breeding, also known as 129X1/SvJ, were obtained from RCC in Fullinsdorf, Switzerland. The resulting colony of α2(H101R) mice were backcrossed to C57BL/6J (Stock No. 000664) mice for at least 5 generations 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 donating laboratory indicates that the Y chromosome was fixed during back-cross 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 males sent to The Jackson Laboratory Repository. One of the 43 markers throughout the genome was segregating with 129, on Chromosome 12.

Allele

Symbol: Gabra2^tm1.1Uru
Name: targeted mutation 1.1, Uwe Rudolph
MGI accession ID: MGI:2183163
Generation method: Targeted
Marker symbol: Gabra2
Marker name: gamma-aminobutyric acid (GABA) A receptor, subunit alpha 2
Chromosome: 5
Strain of origin: 129P2/OlaHsd
Molecular note: This allele was generated by breeding mice carrying Gabra2^tm1Uru with transgenic mice expressing cre recombinase via the EIIa promoter. The floxed neomycin cassette was excised from intron 3, while the point mutation in codon 101 that results in a histidine to arginine substitution remained intact. Western blot analysis showed that the substitution did not affect expression of the protein. Autoradiographs of parasagittal brain sections showed novel diazepam-insensitve sites in regions where the endogenous protein is expressed, indicating the disruption of the drug-binding site.