B6.129(Cg)-Gabra3^tm1.1Uru/J

Stock number: 037211
Product name: α3(H126R)
Research areas: Neurobiology Research

Description

α3(H126R) knock-in mice were generated by incorporating a point mutation in exon 4 of the Gabra3 gene, which encodes a single amino acid substitution (Histidine-126 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 Gabra3 gene encodes the α3 subunit specifically.

This α3(H126R) knock-in model includes a point mutation in exon 4 of the Gabra3 gene that results in replacement of Histidine-126 with Arginine, rendering the α3 subunit to be benzodiazepine-insensitive like the α4 and α6 subunits. 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. The α3(H126R) mice display anxiolytic-like behaviors, similar to wildtype controls, after Diazepam exposure when performing light-dark choice test and elevated plus maze test. Also, no significant changes in Diazepam-induced sedation, motor impairment or anticonvulsant response in α3(H126R) knock-in mice, suggesting that the α3-subunit may not play a role in response to benzodiazepines. However, Diazepam-induced myorelaxation and antihyperalgesia are reduced compared to 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 α3(H126R) knock-in allele was created by introducing a point mutation (Histidine-126 to Arginine) into exon 4 of the Gabra3 gene and a loxP-flanked neomycin resistance (neo) marker placed in the Nco I site downstream of exon 4. The sequence TTC CAC AAT in exon 4 encoding the amino acids FHN (positions 125 to 127) was replaced with TTC CGG AAT encoding the amino acids FRN. Furthermore, a silent mutation for use in restriction fragment length polymorphism analysis was introduced: GGG (threonine at position 150) was replaced by GGT so that a novel Kpn I site (GGTACC) was generated. The construct was electroporated into 129P3/JEms-derived mEMS32 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. 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. 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. Note that the Gabra3 gene is located on the X chromosome.

Allele

Symbol: Gabra3^tm1.1Uru
Name: targeted mutation 1.1, Uwe Rudolph
MGI accession ID: MGI:2183168
Generation method: Targeted
Marker symbol: Gabra3
Marker name: gamma-aminobutyric acid (GABA) A receptor, subunit alpha 3
Chromosome: X
Strain of origin: 129P2/OlaHsd
Molecular note: This allele was generated by breeding mice carrying Gabra3^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 126 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.