Figure 1.
Generation of mice with the Slc26a4 p.H723R mutation.
(A) Targeting scheme. A BAC clone (clone no. bMQ-323G13 Geneservice™) from the 129S7/AB2.2 BAC library containing the mouse Slc26a4 genomic region was used to construct the targeting vector. (1) Restriction map of the wild-type genomic mouse Slc26a4 locus. The expected size of the XbaI restriction fragment was 12.8 kb. (2) Targeting vector (TV) construction. The loxP-flanked neomycin resistance gene (neo) was used as a selection marker during embryonic stem (ES) cell culture. The c.2168 A>G mutation in exon 19 is labeled with a star. (3–4) The targeted locus was introduced, and then, the neo cassette was removed. LA, long arm; SA, short arm. (B) Southern blot analysis of ES cell clones. Genomic DNA from 2 targeted and 2 wild-type clones were digested with XbaI and hybridized with the probe to verify the targeting event. (C) DNA sequencing of Slc26a4+/+ and Slc26a4 tm2Dontuh/tm2Dontuh mice. The electrophoretogram shows the p.H723R mutation. The A to G mutation (arrow) at position 2168 led to the replacement of a histidine (His, H) residue at position 723 with arginine (Arg, R).
Figure 2.
Hearing thresholds (dB SPL) of different frequencies (clicks, 8, 16, and 32 kHz) at 1, 3, 6, and 9 months in mice with different genotypes.
Heterozygous mice (i.e., Slc26a4+/tm2Dontuh), homozygous mice (i.e., Slc26a4tm2Dontuh/tm2Dontuh), and compound heterozygous mice (i.e., Slc26a4tm1Dontuh/tm2Dontuh) showed normal hearing as wild type mice (Slc26a4+/+ ) up to 9 months.
Figure 3.
Comparison of Cochlear morphology in mice with different genotypes at P60.
Compared with cochlear morphology in Slc26a4+/+ mice (A), severe endolymphatic hydrops (dilatation of scala media) and a significant atrophy of the stria vascularis (B), as well as degenerated hair cells (E), were observed in Slc26a4tm1Dontuh/tm1Dontuh mice. In contrast, normal cochlear morphology was revealed in both Slc26a4tm2Dontuh/tm2Dontuh mice (C) and Slc26a4tm1Dontuh/tm2Dontuh mice (D). No degeneration of cochlear hair cells at the basal turn was observed in Slc26a4tm2Dontuh/tm2Dontuh mice (F) and Slc26a4tm1Dontuh/tm2Dontuh mice (G). IHC: inner hair cells; OHC: outer hair cells; RM: Reissner’s membrane; SV: stria vascularis; A, B, C, D: hematoxylin and eosin (H&E) staining; E, F, G: fluorescence confocal microscopy; Bar = 150 µm (A–D) and 20 µm (E–G).
Figure 4.
Comparison of Vestibular morphology in mice with different genotypes at P60 (Slc26a4tm2Dontuh/tm2Dontuh mice: A, B, C, G, H; Slc26a4tm1Dontuh/tm2Dontuh mice: D, E, F, I, J).
Normal morphological findings and amount of otoconia in the vestibule (arrowhead) are shown in both mice (A, B, C, D, E, F). No degeneration of vestibular hair cells in both mice is observed by fluorescence confocal microscopy (G, H). Scanning electron microscopic findings show normal otoconia at the utricle in both mice (I, J). Bar = 50 µm (A–F) and 10 µm (G–H).
Figure 5.
Expression of pendrin and Kcnj10.
(A) Pendrin is normally distributed in the spiral prominence and root cells (stained in green) in Slc26a4+/+ (left), Slc26a4tm2Dontuh/tm2Dontuh (middle), and Slc26a4tm1Dontuh/tm2Dontuh mice (right), indicating that the expression of pendrin is not affected by the p.H723R mutation in mice. (B) Immunoblotting of pendrin expression at P42. Both Slc26a4tm2Dontuh/tm2Dontuh and Slc26a4tm1Dontuh/tm2Dontuh mice expressed pendrin of molecular weight comparable to the wild-type mice, indicating that the glycosylation process remained unaffected in the p.H723R-pendrin. (C) Quantification of pendrin protein expression at P42. The expression levels of pendrin in Slc26a4tm2Dontuh/tm2Dontuh mice and Slc26a4tm1Dontuh/tm2Dontuh mice were 0.89±0.18 and 1.08±0.13, showing no significant difference as compared with 1.00±0.11 in Slc26a4+/+ mice (mean percentage ± SE, n = 3). (D) Quantification of mRNA expression of Kcnj10 at P15 by real-time PCR. Slc26a4tm2Dontuh/tm2Dontuh mice and Slc26a4tm1Dontuh/tm2Dontuh mice did not show significantly different mRNA levels of Kcnj10 as compared with Slc26a4+/+ mice. (E) Quantification of Kcnj10 protein expression at P15 by western blotting. The expression levels of Kcnj10 protein in Slc26a4tm2Dontuh/tm2Dontuh mice and Slc26a4tm1Dontuh/tm2Dontuh mice were 0.90±0.19 and 0.90±0.17, showing no significant difference as compared with 1.00±0.12 in Slc26a4+/+ mice (mean percentage ± SE, n = 3). RC: root cells; SP: spiral prominence; SL: spiral ligament; SV: stria vascularis; Bar = 20 µm.
Figure 6.
Chronological change of hearing thresholds (dB SPL) of different frequencies (clicks, 8, 16, and 32 kHz) in mice with different genotypes.
Neither Slc26a4tm2Dontuh/tm2Dontuh mice nor Slc26a4tm1Dontuh/tm2Dontuh mice had a significantly higher shift in hearing thresholds at any of the frequencies at 30 min or at day 1, 2, 3, 7, and 14 after noise exposure as compared with Slc26a4+/+ mice.
Table 1.
Comparison of phenotypes among mouse strains segregating different Slc26a4 mutations.