Taqi v2

Slack^+/R455H mice were generated in the C57BL/6J mouse strain using CRISPR/Cas9 via the Yale Genome Editing Center. The generation and basic characterization of this mouse model are available in our previous paper.^{19 (link)} Heterozygous (WT/R455H, Slack^+/R455H) mice were used as breeding pairs to obtain WT, heterozygous and homozygous (R455H/R455H, Slack^R455H/R455H) embryos for study. Littermates were labeled and genotyped using gene-specific polymerase chain reaction (PCR) on DNA extracted from tail tissues with primers (Slack forward primer: 5′-ACAGCTGCGGTGAGTTCAGG-3′; Slack reverse primer: 5′-GGGAAGGTTGTCCCAAAGGAGAGC-3′) with standard thermocycler amplification conditions. Following amplification, a restriction cut was performed with the enzyme TaqI-v2 (NEB, R0149S) to distinguish WT (196 and 112 bp products after cut), R455H heterozygous (112, 196, and 308 bp products) and homozygous (308 bp product) alleles. Whenever possible, investigators were blind to the genotype of the mice. GAD67-GFP mice were purchased from Jackson Laboratories (Bar Harbor, MA). For all experiments, male and female littermates were used for each genotype. All experiments were performed in accord with the NIH Guidelines for the Care and Use of Laboratory Animals and approved by Yale University’s Institutional Animal Care and Use Committee (IACUC).