FIGURE 2.

Construction of Arhgef6 knockdown mice. (A) Schematic drawing of the construction method. Targeting sites for Arhgef6 gene disruption. Exon 1 was disrupted at two sites – the first site has a 3 bp deletion, and the second site has a 3 bp insertion and 7 bp deletion, resulting in a frameshift mutation that codes for a truncated protein. (B) Schematic drawing of two isoforms of ARHGEF6 protein. Only isoform 1 was affected by the targeting strategy. (C) Sequencing chromatograms of wildtype, heterozygous, and homozygous female mice. (D) Genotyping results of wildtype, homozygous, and heterozygous mice by PCR. (E) Immunostaining of whole-mount basilar membranes showed strong ARHGEF6 expression in wildtype mice HCs but little expression in knockdown mice HCs at P60. Scale bar: 20 μm. (F) Western blot showed that the ARHGEF6 protein can be detected in spleen, brain, and cochlea lysates from wildtype mice but was nearly undetectable in tissue lysates from Arhgef6 knockdown mice at P30. (G) RT-PCR and qRT-PCR showed the expression of Arhgef6 isoform1, isoform2, and total Arhgef6 at stages P3 and P14 in wildtype and knockdown mice.