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. 2018 Jul 11;38(28):6267–6282. doi: 10.1523/JNEUROSCI.0515-18.2018

Figure 1.

Figure 1.

Removal of Rosaβgeo insertion allows re-expression of βIV spectrin. A, Partial genomic map of the Sptbn4 locus and the location of the gene trap Rosa βgeo* insertion, which is removed by Cre-mediated recombination. Red arrowheads represent location of primers that were used for qRT-PCR amplification of Sptbn4 transcripts. B, PCR amplification of genomic tail DNA isolated from wild-type (+/+), heterozygous (Sptbn4geo/+), and homozygous floxed (Sptbn4geo) mice. C–L, Immunostaining of teased SN fibers (C–G) and SCs (H–L) from control (+/+) (C, H), Sptbn4geo mutant (D, I), and tamoxifen-injected 2-month-old, 5-month-old, and 8-month-old actin-CreER;Sptbn4geo mice after 1 mpi (E–G, J–L, Sptbn4res) with antibodies against nodal βIV spectrin (red) and paranodal Caspr (green). Red arrowheads point to βIV spectrin-positive nodes and white arrowheads indicate βIV spectrin-negative nodes. M, N, Percentage of βIV spectrin-positive nodes in SNs (M) and SCs (N) of control, Sptbn4geo mutant, and Sptbn4res rescue animals depicted in C–L. Data are represented as mean ± SEM. n = 3 mice per genotype, with ≥100 and 200 nodes/animal for SNs and SCs, respectively. ***p < 0.001, one-way ANOVA, Bonferroni's post hoc analysis. Scale bar, 4 μm. O–Q, Immunoblot analysis of SN (O), SC (P), and brain (Q) lysates from control, Sptbn4geo mutant, and Sptbn4res rescue animals 1 mpi with antibodies against βIV spectrin (top) and tubulin (bottom). R, S, qPCR analysis of relative mRNA quantity in DRG (R) and SC (S) samples from control, Sptbn4geo mutant, and Sptbn4res rescue animals 1 mpi using primers specific to various exon/intron junctions of the Sptbn4 locus (exons 17/18, 20/21, and 31/32, depicted in the schematic in A as 1-2, 3-4, and 5-6, respectively). Data are represented as mean ± SEM. n = 4–7 mice per genotype. ***p < 0.001, one-way ANOVA, Bonferroni's post hoc analysis. T, Schematic representing the design of rescue strategies. Tamoxifen injection times in actin-CreER;Sptbn4geo animals and the timeline for phenotypic analysis of the two rescue groups.