(A) Construct design and validation of A1783V variant transcript expression. Note that this line was developed by Ana Mingorance (Chief Development Officer of the Loulou Foundation) and is available at JAX (sock # 026133). Ai, schematic shows loxP sites flanking wild type exon 26 followed by an edited version of exon 26 that contains the human A1783V pathological variant (ΔE26). When Cre recombinase is expressed, wild type exon 26 is removed, thus allowing transcription of ΔE26. Aii, Agarose gel shows detectable levels of Scn1a transcript (expected size of 831 bp) in brainstem tissue isolated from each genotype (primers span between exon 25 and 26, including residue 1783 of exon 26). Water was used as a no template negative control. Aiii, PCR products were sequenced to confirm that transcript containing A1783V is detectable in 40% of samples from Scn1aΔE26 tissue but was not detectable in samples from Slc32a1cre/+ and Scn1afl/+ control tissue. (B–C), fluorescent in situ hybridization (RNAScope) was performed to characterize expression of Scn1a transcript in inhibitory (Slc32a1+, Slc32a1) and glutamatergic (Vglut2+, Slc17a6) neurons in the RTN region in brainstem sections from control and Scn1aΔE26mice. (B) brainstem sections from Slc32a1cre/+ and Scn1aΔE26 mice containing the RTN show Scn1a labeling (green puncta) of both Vgat+ and Vglut2 +neurons. (C), summary data show Scn1a transcript expression (normalized to cell size) in Vgat+ and Vglut2 +RTN neurons from each genotype; channel transcript was reduced in Vgat+ cells from Scn1aΔE26 mice (0.43 ± 0.7 mRNA/area, n = 94 cells) compared to control (0.73 ± 0.9 mRNA/area, n = 82 cells) (p<0.05), whereas Vglut2 +cells showed low channel transcript across both genotypes. (D–E) Scn1aΔE26 mice did not show any obvious differences gross morphology (A) body weight (D) or temperature (E) compared to age-matched litter mate control mice. (F) (Figure 1—source data 1), survival curve shows that control mice (n = 57) survive to adulthood (30 days postnatal) while Scn1aΔE26 mice (n = 41) die prematurely starting at 9 days postnatal and reaching 100% lethality by 25 days (χ2 = 63.9, p<0.0001). These results were compared using a two-way ANOVA and Sidak multiple comparison test. *, p<0.05; ***p<0.001; ****p<0.0001.