Generation of appa−/− and analysis of appa−/−appb−/− double mutant zebrafish. (A) Schematic outline of the appa gene with exons (black box) and UTR regions (white box). sgRNA used to target exon 2 with protospacer adjacent motif (PAM) in red and the sgRNA target sequence underlined. (B) Sanger sequencing chromatogram of the exon 2 region targeted in wild-type zebrafish. The appa−/− mutant sequence is given below with dash indicating deleted nucleotides. (C) Schematic drawing of the wild-type Appa protein (738 aa) with epitopes of antibodies (dotted squares) used above and the hypothetical truncated Appa (109 aa) protein produced in appa mutant below. (D) qPCR quantification of appa and appb mRNA levels in wild-type and appa−/−appb−/− mutants at 24 hpf. (E) Western blot of 3 dpf whole larvae zebrafish with antibodies against 22C11 and App (Y188). Alpha-tubulin is used as loading control. Blots cropped from the same original gel and grouped. Quantification of band intensity are shown relative to control. Data are reported as mean ± SD. ** ρ < 0.05, **** ρ < 0.001. qPCR n = 5, WB n = 3. SP signal peptide, E1 extracellular domain, ED extension domain, AcD acidic domain, E2 extracellular domain 2, JMR juxtamembrane region, Aβ amyloid beta, AICD amyloid intracellular domain.
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