Intellectual disability-associated UNC80 mutations reveal inter-subunit interaction and dendritic function of the NALCN channel complex

摘要

The design of knockout (KO) using the CRISPR technique to delete exon 3. Exon 3 sequence is in capital and the surrounding introns are in lower case. The 5′ and 3′ target sequences including the PAM motif (XGG) against which the two CRISPR sgRNAs targeted are underlined. Deleted sequences including exon 3 and part of the surrounding introns are shaded. Deletion of exon 3 (total of 157 nucleotides) leads to truncation after V47. The codon encoding R51 (CGA) corresponding to the residue mutated to a stop codon found in human patients are in red. PCR primers used for genotyping in are in italic and boxed. Genotyping PCR products from WT (+/+), heterozygote (+/−), and homozygous KO (−/−) pups using primers in . Total brain proteins from +/+ and −/− were blotted with anti-UNC80 (upper), anti-NALCN (middle), or anti-UNC79 (lower) antibody. Representative appearances of WT and KO P0 pup. For ( ) and ( ), three or more independent repeats were performed with similar results. For apnea phenotype in the KO, see Supplementary Movie  . Source data are provided as a .