Fig. 4.

Analysis of the Nox3^het–3J allele. a Sanger sequencing-based confirmation of the Nox3^het–3J mutation identified by HTP sequencing. The mutation consists of a G > A transition at the −1 position of the splice acceptor site just prior to exon 2. b RT-PCR analysis of C57BL/6J and Nox3^het–3J/Nox3^het–3J inner-ear RNA with 1F/2R and 1F/4R primer pairs. Lane 1, 100-bp ladder; lane 2, wild-type RNA; lane 3, negative reverse transcriptase (−RT) control; lane 4, Nox3^het–3J/Nox3^het–3J RNA; lane 5, −RT control; lane 6, no template control. c Models of wild-type (C57BL/6J) and mutant (C57BL/6J-Nox3^het–3J/Nox3^het–3J) Nox3 transcripts. Red numbered rectangles, exons; asterisk, mutation; C, cryptic splice acceptor; blue rectangle, intronic sequence incorporated into the mutant transcript. d Whole-mount preparation of cleared temporal bones from adult Nox3^het–3J/+ and Nox3^het–3J/Nox3^het–3J mice. Note the presence of otoconia (arrows) in the heterozygotes and their absence in mutant homozygotes