Generation and characterization of Tmem150c mouse models. (A) Left: Schematic representation of Tmem150c locus of the knockout first mouse (Ttn3LacZ), named here Tmem150cLacZ, generated by the trans-National Institutes of Health Mouse initiative knockout Mouse project (https://www.komp.org) containing a LacZ cassette and a neomycin cassette with a stop codon inserted between exons 5 and 6 and resulting in a frame shift. Right: Representative gels showing PCR results for neomycin cassette, LacZ cassette, and WT bands. A and B primers (in blue) used for the amplification of the WT band. Genomic DNA from ear biopsies from WT, heterozygous, and homozygous mice were analyzed. M: DNA marker. (B) β-galactosidase staining (blue) of epididymis (positive tissue control) and DRG of Tmem150cLacZ/LacZ mice. Note the unexpected lack of staining in the DRGs of Tmem150cLacz/LacZ mice. WT tissues used as negative control. Scale bar = 20 μm. (C) Top: Schematic representation of Tmem150c cDNA (source ensemble Tmem150c-201) containing 8 exons (1–8) with the start codon located in exon 2 (2) and the stop codon located in exon 8 (8). Bottom: RT-PCR performed on cDNA prepared from tissues of WT mice and Tmem150cLacz/LacZ mice with the blue line indicating amplicon covering exon 4–6 (the targeted area of Tmem150cLacZ allele) and the green line indicating amplicon covering the end of exon 6 to the beginning of exon 8. Note the presence of unexpected bands in DRG of the Tmem150cLacz/LacZ mice but the absence of bands in Epi and Li as expected. Hprt1 (housekeeping gene) is used as positive control for all tissues. Epi, epididymis (positive control); Li, liver (negative control). (D) Schematic representation of Tmem150c null allele generation using CRISPR/Cas9 technology: deletion of nucleotide sequence between the end of intron 1–2 and the beginning of intron 5–6. WT allele and null allele are shown: exon1 (E1) encodes the 5′ UTR and E8 the 3′ UTR (white box). Black box: coding sequence. (source ensemble ENSMUSG0000005064017). Red arrows indicate the location of gRNA sequences used for CRISPR/Cas9. (E) PCR performed on genomic DNA from ear biopsies from WT, heterozygous, and homozygous mice are shown. The WT amplicon is represented by the green line in the scheme covering E3 and E4 (179 bp). The null allele amplicon is represented by the blue line producing an 886 bp fragment when the nucleotide sequence between intron 1–2 and intron 5–6 (3,039 bp in WT) is deleted as in the null allele. Note that this PCR fragment could amplify a 3,039 bp fragment in WT mice (see blue line), but this is not possible using the chosen PCR conditions. M: DNA marker. (F) TMEM150C protein expression using Western blot showing absence of TMEM150C protein in tissues from Tmem150c−/− mice. Epididymis (Epi, positive control) and liver (Li, negative control). β-actin was used as loading control. (G) Immunostaining performed on DRGs from WT and Tmem150c−/− mice using an antibody directed against the C-terminal part of TMEM150C (same as in F). Note that TMEM150C antibody labeled neurons in WT, but also in Tmem150c−/− mice. NF200 was used to label myelinated neurons. Scale bar = 50 μm. Source data are available for this figure: SourceData F2.