STOCK Gabra5^tm1.1Uru/J

Stock number: 037212
Product name: α5(H105R)
Research areas: Neurobiology Research

Description

α5(H105R) knock-in mice were generated by incorporating a point mutation in exon 4 of the Gabra5 gene, which encodes a single amino acid substitution (Histidine-105 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 Gabra5 gene encodes the α5 subunit specifically.

This α5(H105R) knock-in model includes a point mutation in exon 4 of the Gabra5 gene that results in replacement of Histidine-105 with Arginine, rendering the α5 subunit to be benzodiazepine-insensitive like the α4 and α6 sunbunits. The donating investigator reports that these knock-in mice do not demonstrate an overt phenotype. The α5(H105) mice displayed anxiolytic, sedative and anticonvulsant responses, after Diazepam exposure, similar to wildtype controls. However, α5(H105) mice did demonstrate changes in grasping reflex as assessed by the horizontal wire test. Another striking difference in the α5(H105) model, is that these knock-in mice have a significant reduction in the number of α5 subunits in hippocampal pyramidal cells compared with wild type. Using a trace fear conditioning task, the donating investigator found that the α5(H105) mice demonstrated a significant difference in freezing-time 48 hours after paired-association was initially presented. This suggests that the α5(H105) mice may demonstrate a dysfunction in temporal processing of associative learning. 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 α5(H105R) knock-in allele was created by introducing a point mutation (Histidine-105 to Arginine) into exon 4 of the Gabra5 gene and a loxP-flanked neomycin resistance marker in intron 4. The construct was electroporated into 129X1/SvJ-derived RW-4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into 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. The resulting colony of α5(H105R) mice were maintained on the 129/Sv background by the donating laboratory (see SNP note below). As per the donating investigator the 129/Sv mice utilized for breeding, also known as 129X1/SvJ, were obtained from RCC in Fullinsdorf, Switzerland. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize 129X1/SvJ oocytes (Stock No. 000691). Of note, the Gabra5 gene is close to the pink-eyed dilution locus (Oca2).

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. Eight of the 43 markers throughout the genome were segregating with an unknown source, suggesting the mice sent to The Jackson Laboratory Repository were on a mixed genetic background.

Allele

Symbol: Gabra5^tm1.1Uru
Name: targeted mutation 1.1, Uwe Rudolph
MGI accession ID: MGI:2183170
Generation method: Targeted
Marker symbol: Gabra5
Marker name: gamma-aminobutyric acid (GABA) A receptor, subunit alpha 5
Chromosome: 7
Strain of origin: 129X1/SvJ
Molecular note: This allele was generated by breeding mice carrying Gabra5^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 105 that results in a histidine to arginine substitution remained intact. Western blot analysis showed that the expression of the encoded protein was decreased by approximately 20% in homozygous mutant mice. Autoradiographs showed novel diazepam-insensitve sites in regions where the endogenous protein is expressed, indicating the disruption of the drug-binding site.