Extended Data Fig. 7: Generation and validation of an Slc45a4 KO mouse.
From: SLC45A4 is a pain gene encoding a neuronal polyamine transporter
a, Schematic of the Slc45a4 locus outlining the 8 exons, the start and stop sites, the CRISPR-Cas9 KO region targeted with guide RNAs (gRNA). The illustration also highlights where the genotyping and qPCR primers target and the RNAscope probe. b, Example genotyping gel of WT, HET and KO mice. PCR amplified WT band 652 bp, KO band 468 bp. This was performed once for all mice (original gel in source data). c, Images of Slc45a4 KO mice at different ages, at about 6 weeks mice develop a salt and pepper/white speckled hair. This is transient and returns to normal by week 12. d, qPCR was used to analyse the expression of Slc45a4 mRNA in WT HET and KO mice, in DRG, Spinal cord and Brain respectively. Heterozygous mice have significantly reduced Slc45a4 mRNA compared to WT, and there is a complete absence of expression in KO mice across tissues (n = 4 mice per group) e, Analysis of Slc45a4 mRNA signal intensity in DRG sections from WT, HET and KO mice (WT n = 1349 cells from 4 mice, HET n = 1452 cells from 3 mice, KO n = 951 cells from 3 mice). Threshold set using a negative control probe. d and e, one-way ANOVA with post hoc Tukey test, d, DRG **** P < 0.0001. Spinal cord * P = 0.026, ** P = 0.0018, **** P < 0.0001. Brain * P = 0.018, *** P = 0.0008, e, **** P < 0.0001). Data mean ± s.e.m.
