(A) The Tspan6 KO mouse was generated by insertion of a neomycin cassette in the exon 2 of the Tspan6 gene. Right panel show a representative agarose gel electrophoresis with the PCR products amplified with specific primers (a, b and c, shown by arrows in the left panel). (B) RNA was extracted from Tspan6 KO and WT animals. Primers were designed between exon 1 and 3 (WT-specific primers), and exon 4 and exon 5 (primers downstream insertion). (C) Real time semi-quantitative PCR shows no RNA amplification between exon 1 and 3 in Tspan6 KO mice due to the insertion of the neomycin cassette. (D) RNA amplification downstream the insertion is reduced in Tspan6 KO mice (0.35± 0.01 mean fold change compared to WT) suggesting RNA degradation. Histogram shows mean (±S.E.M) fold changes normalized against WT expression, using either WT-specific primers (C) or primers downstream the insertion (D). Two housekeeping genes (Actin and GAPDH) were used for the normalization of the expression. (E) Neuronal lysates from cortical primary cultures from Tspan6 WT, heterozygous and KO mice show the absence of Tspan6 protein in the KO condition. (F) RNA scope shows expression of Tspan6 RNA in the pyramidal layer of the hippocampus and granule cells from the dentate gyrus. First panel is a general view of the hippocampus (scale bar = 100μm). Panels 1 to 5 show box section in higher magnification (scale bar = 10μm).White dots are Tspan6 RNA molecules, synaptophysin RNA is stained in red and DAPI in blue.

https://doi.org/10.1371/journal.pone.0187179.g001