2.3. Generation and validation of the GABAA-δ subunit knockout mice

To understand what behaviors could be affected by reducing GABA_A-δ subunit function, we used a refined strategy for conditional gene inactivation that relies on the DNA recombinase Cre and its recognition loxP sites. Cre-loxP system is a widely used powerful technology for mammalian gene editing. This system has advantages which is very simple manipulation and do not require additional factors for efficient recombination (Nagy, 2000; Kim et al., 2018). In this study, floxed Gabrd mice (Lee and Maguire, 2014; JAX stock #023836), were crossed with CMV-Cre mice obtained from Jackson Laboratory (stock #006054) to generate mice deficient in the Gabrd gene. CMV-cre is a Cre-driver strain that express Cre recombination allowing deletion of loxP-flanked genes all tissues (Schwenk et al., 1995). Mouse genotypes from tail biopsies were determined using real-time PCR with specific probes designed for each gene: floxed, wild-type and Cre (Transnetyx, Cordova, TN). Mice of both sexes were used for experiments from 8–12 weeks of age (25–35 g).

Crossing Gabrd floxed^(+/+) mice with CMV-cre^(+/+) homozygous mice produced an F1 generation with the genotype Gabrd^+/− CMV-cre⁺. Subsequently, Gabrd^+/− CMV-cre⁺ females and Gabrd^+/+ males were bred, leading to an F2 generation with a range of genotypes. In this manuscript we refer to the offspring which have an intact δ-subunit gene on both alleles as control mice (Gabrd^+/+ CMV-cre⁻ and Gabrd^+/− CMV-cre⁻). We refer to the offspring which have an intact δ-subunit gene on one allele as δ HET (Gabrd^+/− CMV-cre⁺). We refer to the offspring which have the δ-subunit gene deleted from both alleles as δ KO (Gabrd^−/− CMV-cre⁺).