The THIK‐1 gene (kcnk13) was disrupted by MRC Harwell by using CRISPR/Cas9 to insert a single nucleotide into the wild‐type DNA sequence (Bradley et al., 2012 (link); Brown & Moore, 2012 (link); Pettitt et al., 2009 (link)). Insertion resulted in a frameshift mutation in the codon of for amino acid 14. This resulted in a premature stop codon after amino acid 68. The mice were maintained as homozygotes on a C57BL/6 background. To confirm the genotype of the mice Taqman MGB Allelic Discrimination genotyping assays were designed using Primer Express 3.0.1 (Applied Biosystems). Taqman MGB probes were purchased from ThermoFisher & primers were purchased from Sigma Aldrich.
Primer/probe nameSequence (5′–3′)
MmKCNK13 SNP genotyping FPGGTCGGCAGAGCACATCCT
MmKCNK13 SNP genotyping RPCTGCAACTCCTGCGCTAGCT
MmKCNK13 WT SNP genotyping probeFAM‐CACCTGAACGAGGAC‐MGB
MmKCNK13 KO SNP genotyping probeVIC‐CACCTGAATCGAGGAC‐MGB
Lysates were prepared from ear snips using Extract‐N‐Amp Tissue PCR Kit from Sigma (XNAT2R). qPCR was run in 384 plates with the following thermocycler conditions: 60°C × 30 s, 95°C × 10 min, then 40 cycles at 95°C × 15 s, 60°C × 1 min, lastly one cycle at 60°C × 30 s.
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