B6;CBA-Gabrd^em1Menn/J

Stock number: 032620
Product name: δ* KI
Research areas: Cell Biology Research, Neurobiology Research

Description

δ* KI is a knock-in allele with a T269Y mutation in Gabrd exon 8 that renders δ-containing receptor populations insensitive to picrotoxin (PTX). These mice allow chemogenetic isolation of δ-containing GABA_A receptors, and may be useful for neurological studies of receptor, ion channel and synapse biology, neurotransmitters, and glutamate/GABA transmission.

Detailed description

γ-aminobutyric acid type A receptors (GABA_AR) are ionotropic receptors and ligand-gated ion channels that play key roles in transient and tonic inhibition. GABA_AR-mediated inhibition sculpts neuronal activity and regulates key behaviors. Gabrd encodes the δ subunit of the GABA_AR family. Receptors containing the δ subunit have been implicated in consciousness, mood disorders, schizophrenia and epilepsy.

δ* KI is a CRISPR/cas9-made knock-in allele with a T269Y mutation in Gabrd exon 8 that renders δ-containing receptor populations insensitive to the GABA_AR antagonist picrotoxin (PTX), while having little effect on basal receptor function. Specifically, the donating investigator reports δ KI homozygotes may exhibit a shift in steady state sensitivity to GABA (1.5 micromolar to 3.5 micromolar).

Unless noted otherwise, the phenotype of mice homozygous for the δ* KI allele are described below. δ* KI homozygotes are viable and fertile, with no reported gross phenotypic abnormalities. Hippocampal tissues from δ* KI and wildtype mice exhibit no differences in δ subunit protein levels. In the absence of PTX, δ* KI and wildtype brain cell slices show no detectable differences in evoked transmission in dentate granule cells (DGCs). Following PTX treatment in brain cell slices, evoked IPSCs were abolished in wildtype DGCs, whereas a portion of evoked IPSCs persist in δ* KI DGCs (exhibiting much slower decay kinetics). Similar results were observed in other δ-containing GABA_AR cell types (e.g., thalamocortical neurons). [Sun et al. 2018 J Neurosci 38:8128]
The donating investigator reports that more extensive behavioral characterization has not been performed to date (November 2018).

Mice heterozygous for the δ* KI allele are viable and fertile with normal breeding, and no reported gross phenotypic abnormalities. The donating investigator reports that although limited electrophysiological characterization for δ* KI heterozygotes has been performed to date (November 2018), the heterozygous mice have nearly complete PTX sensitivity, similar to wildtype.

Development

The δ* KI allele was created in the Washington University Transgenic Vectors Core and the laboratories of Drs. Joe Henry Steinbach and Steven J. Mennerick (Washington University School of Medicine in St. Louis) using CRISPR/cas9 endonuclease-mediated genome editing. First, single guide RNAs (sgRNAs ; gctgacaatgaccacgctCa) were designed to target sequences in exon 8 of the gamma-aminobutyric acid (GABA) A receptor, subunit delta locus (Gabrd) on chromosome 4. Single-stranded oligodeoxynucleotide (ssODN) donor DNAs encoding the Gabrd T269Y mutation were obtained (tgaagaactcccctttccctggaacccagccctgttggctagggtaaggaggagagggtaggacttcatcgctgaccatctactcccaccaggcatcTACactgtgctgacaatgaccacgctTatggtgtgcccgctcctccctgccacgggcttcagctatcaaagctctggatgtgtatttttggatctgctacg). The sgRNAs, ssODN donor DNAs and S. pyogenes CRISPR/cas9 mRNA were injected into B6CBAF1/J fertilized eggs. Founder mice were screened by PCR and sequencing of the Gabrd exon 8 region. This revealed the δ* KI allele had the T269Y mutation in exon 8, as well as an unanticipated C-to-A nucleotide change in intron 7 (15 nucleotides upstream of the desired mutation). Of note, predictive software showed this C-to-A mutation imparts no expectation for altered splicing. Founder male #180-9 was bred to wildtype C57BL/6J mice for germline transmission. The donating investigator reports the δ* KI colony was then maintained on C57BL/6J;CBA/J genetic background by breeding heterozygous mice together, and was not characterized for the presence/absence of off-target mutations. In 2018, males with agouti coat color were sent to The Jackson Laboratory Repository. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Gabrd^em1Menn
Name: endonuclease-mediated mutation 1, Steven Mennerick
MGI accession ID: MGI:6209489
Generation method: Endonuclease-mediated
Attributes: Not Specified
Synonyms: delta* KI
Marker symbol: Gabrd
Marker name: gamma-aminobutyric acid (GABA) A receptor, subunit delta
Marker synonyms: EIG10; EJM7; GABAA-RD; GABAARdelta; GEFSP5
Chromosome: 4
Strain of origin: (C57BL/6 x CBA)F1
Molecular note: Codon 269 was targeted with single-stranded oligodeoxynucleotide (ssODN) donor DNAs, sgRNAs and Cas9 using CRISPR/Cas9 technology. All components were microinjected into the cytoplasm and pronuclei of fertilized eggs. The resulting p.T269Y (p.Thr269Tyr) mutation renders the receptor picrotoxin (PTX) resistant without affecting basal receptor function. All founder mice for this allele harbored an additional mutation in intron 7, 15 nt upstream of the targeted mutation. In the chosen mouse line this is a C>A substitution that is predicted to have no effect on splicing.