FIGURE 1.

Generation of Scn9a^R185H mutant mice and Scn9a transcript expression in wild-type (wt) and mutant mice. (A) Scheme for Scn9a targeting strategy. The sgRNA86 guide RNA and protospacer adjacent motif (PAM) are shown. The ssODN contained three basic mutations including two silent mutations and one-point mutation. The first silence mutation, C > T was PAM mutation designed to avoid Cas9 recut. The second mutation, C > T, was designed for BspHI enzyme restriction site for genotyping. The third G > A mutation was designed to get the R185H found in human patients. (B) Mutant allele sequence characterized through Sanger sequencing of F0 founder DNA. The silence-mutated nucleotides are indicated in green and targeted point mutant nucleotide in red. (C) Scn9a micro-RNA (mRNA) expression in dorsal root ganglia (DRG) and spinal cord, of wt and Scn9a^R185H mutant mice of both sexes. There was no significant effect of genotype. DRG. females: wt n = 4; Het n = 4; Homo n = 3; males: wt n = 5; Het n = 4; Homo n = 4; Spinal cord. Females: wt n = 5; Het n = 5; Homo n = 6; males: wt n = 5; Het n = 4; Homo n = 5. Scn9a transcript expression was normalized to Hprt expression. Results are shown as means ± SEM. Two-tailed unpaired t-test for wt allele transcript expression in wt mice vs. mutant allele transcript expression in homozygous mutant mice. Refer to Supplementary Table 5 for statistics.