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. 2021 Dec 20;145(9):3187–3202. doi: 10.1093/brain/awab467

Figure 1.

Figure 1

Generation and validation of NHE6-null rats. (A) Two single guide RNAs (sgRNA) were inserted into exon 7 of the endogenous Slc9a6 gene. (B) Schematic representation of the targeted Slc9a6 locus harbouring the 2-bp (TT) insertion. The insertion generated a premature stop codon. (C) Sequences of wild-type (WT) and NHE6-null rats. Genomic DNA sequence, isolated from rat tail biopsy, shows the sequence in the wild-type rat and the 2-bp insertion causing a premature stop codon in NHE6. (D) The mRNA level of NHE6 in wild-type and NHE6-null rat brain was measured by quantitative real-time PCR. Two different sets of primers were used to validate the mRNA level. The mRNA level was normalized against the reference gene. Two-tailed unpaired t-test with Welch’s correction was used (P < 0.0001 for both primer sets, n = 3 for each genotype). (E) Absence of NHE6 protein in NHE6-null rat brain. Immunoprecipitation (IP) of NHE6 in the whole brain lysates from wild-type and NHE6-null rats validates the absence of NHE6 protein. Actin was used as a loading control. IgG Heavy chain was detected at ∼50 kDa after IP with NHE6 antibody. (F) Validation of NHE6 protein absence by immunofluorescence. Brain sections from wild-type and NHE6-null rats at 2 months were stained with NHE6 antibody. Scale bar = 500 µm.