Targeting strategy for disruption of the <i>Trpml3</i> gene.

<p>A, Transmembrane-spanning domains are depicted as grey bars and numbered from 1–6. The orange frame indicates the part of the TRPML3 protein that is encoded by exon 11, which will be deleted (pore loop and TM6). Exons are shown as black and orange bars on the schematic genomic map below. B, Shown are the targeting vector and the targeted allele after homologous recombination. The blue bar represents the PGK promoter-driven <i>neo^R</i> expression cassette, which was used for positive selection. The DTA cassette, used for negative selection, is shown in green. The black and red arrowheads symbolize position and orientation of <i>FRT</i> and <i>loxP</i> sites. C, Targeted allele after Flp site-specific recombination in ES cells, resulting in excision of <i>neo^R</i> cassette, and leaving one FRT site behind. D, Excision of exon 11 using Cre site-specific recombinase, resulting in disruption of <i>Trpml3</i> gene.</p

Rotarod experiments.

<p>The average latencies to fall ± SEM (in sec) are shown for 3-month-old <i>Hprt^Cre/+;Trpml3^+/+</i> (n = 8) and <i>Hprt^Cre/+;Trpml3 ^Δ/Δ</i> (n = 8) mice. The experiment was executed over a time range of 6 days. The day 6 experiment was performed in the dark to exclude compensation via visual cues. The difference between genotypes was not statistically significant at any given time point (p>0.05, one-way ANOVA, followed by Tukey's post test).</p

ABR measurements.

<p>A, Graph shows representative ABR waveforms of 3-week-old <i>Hprt^Cre/+; Trpml3^+/+</i> and <i>Hprt^Cre/+; Trpml3 ^Δ/Δ</i> mice in response to a click stimulus. ABRs were recorded at sound stimulation intensities of 25–70 dB. ABR waves I–V are indicated above the peaks. Red arrow highlights the hearing threshold, which is at 35 dB in this representative example pair. B, Shown are ABR thresholds (mean values ± SEM) to click, 8-, 16-, and 32 kHz stimuli of <i>Hprt^Cre/+;Trpml3^+/+</i> (n = 6), <i>Hprt^Cre/+;Trpml3^Δ/+</i> (n = 6), and <i>Hprt^Cre/+;Trpml3 ^Δ/Δ</i> (n = 6) and C, of <i>Math1-CreER^Cre/+;Trpml3^+/+</i> (n = 3), <i>Math1-CreER^Cre/+;Trpml3^Δ/+</i> (n = 3), and <i>Math1-CreER^Cre/+;Trpml3 ^Δ/Δ</i> (n = 3), respectively. Statistical comparisons of means of different genotypes were made using one-way ANOVA followed by Tukey's post test; p>0.05. D, ABR threshold shifts of 3-month-old <i>Hprt^Cre/+;Trpml3^+/+</i> (n = 7) and <i>Hprt^Cre/+;Trpml3 ^Δ/Δ</i> (n = 7) 1 week after the acoustic overexposure of 125 dB SPL at 4 kHz for 4 hr. Shown are mean values ± SEM. No significant differences were observed between genotypes (p>0.05, one-way ANOVA, followed by Tukey's post test).</p