Fig. 1: Validation of the Tubb4b^−/− murine model.

A Schematic illustrating the mutation in Tubb4b generated by CRISPR-Cas9. gRNA sequences targeting the introns are shown in grey, with the protospacer adjacent motif (PAM) sequence underlined. Deletion of exons 2 and 3 led to changes to the amino acid sequence (grey) and introduces a premature stop codon (grey asterisk). Arrows in exons 1 and 4 indicate the locations of forward and reverse primers used for RT-PCR. B Left panel: Immunoblot of retinal lysates from P30 animals probed with TUBB4 antibody. The blots were also probed for GAPDH, a housekeeping protein serving as a control for protein loading. Molecular weights in kDa are indicated on the left. Right panel: RT-PCR analysis of the Tubb4b and Tubb4a mRNA extracted from retinal tissue, followed by agarose gel electrophoresis. Markers in base pairs (bp) are indicated on the left. Expected sizes for Tubb4b and Tubb4a are 434 bp and 619 bp, respectively. C Immunoblot of cochlear lysates from 1-month-old Tubb4b^−/− and wildtype littermates, probed with TUBB4 and GAPDH antibodies. All experiments were replicated at least three times with littermates as controls (+/+). D The number of pups obtained from Tubb4b^+/− crosses followed Mendelian ratios (total number of animals counted 271). Expected numbers were calculated as (total number of animals counted) * expected ratio (0.25 for +/+, − /−; 0.5 for +/−). The percentage of pups born for each genotype are indicated in the brackets. E Image of 1-month-old male Tubb4b^−/− and control littermate. F Whole bodyweight of Tubb4b^−/− and control littermates at 1 month. Data are presented as mean ± SEM. ***P value ≤ 0.001.