Fig. 5
KLHL17/AF knockout mice show impairments of dendritic spinogenesis and functional synapse formation. a Generation of KLHL17/AF knockout mice by CRISPR/Cas9 technology. Schematic diagram of genomic editing using CRISPR/Cas9 gRNA to delete the klhl17 gene. Small arrowheads indicate the position of primers for genomic PCR in (b). b Genomic PCR for genotyping. The sizes of PCR products were 364 bp for the WT allele and 500 bp for the KO allele. c Total cell extracts from cultured hippocampal neurons of different genotypes were collected at 18 DIV for immunoblotting. HSP90 was used as an internal control. d–f Klhl17+/– and Klhl17–/– mice exhibit impaired dendritic spinogenesis. Cultured hippocampal neurons of different genotypes were transfected with GFP-actin at 12 DIV and immunostained at 18 DIV. Cultured neurons from Klhl17+/– and Klhl17–/– mice exhibit narrow spine heads, but no change in spine length or spine density. d Representative images. e Quantification of protrusion density. f Quantification of width and length of dendritic protrusions. g, h Klhl17+/– mice exhibit a reduced number of functional synapses. Cultured hippocampal neurons of Klhl17+/+ and Klhl17+/– mice were immunostained for PSD95 and vGLUT1 at 18 DIV. g Representative images. h Quantification of overlap coefficients. Samples were randomly collected from two independent experiments without knowing the genotypes. N the number of analyzed dendrites; n, the number of analyzed dendritic protrusions. Data represent the mean plus SEM. ***P < 0.001; ns not significant. Scale bar: d 5 μm, g 1 μm. One-way ANOVA (e), unpaired t-test (h), Kolmogorov–Smirnov test for cumulative probability (f)