Fig. 2: Mutant mouse model design and validation.

A Left, schematic showing knockout design for Klhdc7b^RegnΔ/Δ mice. The whole Klhdc7b gene was excised from the mouse genome and replaced with a beta-galatosidase (LacZ) expression cassette for visualization. Right, knockout design for Klhdc7b^IMPC-/- mice. CRISPR/Cas9 was used to create a 1258 base pair deletion creating a truncating premature stop codon and a null allele. The location of the putative short isoform is shown with a smaller box above or below the Klhdc7b long isoform in both schematics. B LacZ expression in wholemount preparations of the organ of Corti of heterozygous (Klhdc7b^Regn+/Δ) and knockout (Klhdc7b^RegnΔ/Δ) mice (males age 10 weeks). Some OHC loss C 40x confocal images showing RNAscope Klhdc7b probes for the L and L + S transcripts and hair cell marker MYO7A immunofluorescence of Klhdc7b^Regn+/+ mouse organ of Corti. DAPI stains nuclei. Outlines of MYO7A staining are shown overlaid in white on individual probe channels to delineate hair cells. D As C, but in Klhdc7b^RegnΔ/Δ mouse organ of Corti. For C, D, mice are females age 8 weeks. E KLHDC7B immunofluorescence of Klhdc7b^Regn+/+(top) and Klhdc7b^RegnΔ/Δ mice (bottom). Mice are 13–14 weeks, WT is male and KO is female. F Confocal images showing cochleae from Klhdc7b^IMPC+/- mouse organ of Corti at postnatal day 19. Sections have been stained for KLHDC7B (white), MYO7A (red) and DAPI (blue) (female postnatal day 19). G Same as F, in Klhdc7b^IMPC-/- mouse organ of Corti (female at postnatal day 19).