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A 1.3-kb Acc I-Bam Hl fragment and a 6-kb Eco RI fragment isolated from a J1 genomic DNA library were used as short and long homologous sequences of a targeting vector, respectively. The pgk-neo cassette flanked by a pair of loxP sequences was used for positive selections, and the diphtheria toxin A-chain gene cassette without a polyadenylation site was used for negative selection. The Not I-linearized targeting vector was introduced into J1 ES cells derived from 129/sv mice by electroporation and cells were selected with G418 (17). 6. An OCTA26 probe (GATCAGTACTAAT TAGCATTATAAAG) was used.
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0345256279
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Mice were anesthetized with sodium pentobarbital (70 mg/kg) and maintained in a headholder within an acoustically and electrically insulated test room. Needle electrodes were placed on the tympanic bulla (positive lead) and scalp vertex (negative lead). Acoustic stimuli evoked by a dick were delivered and the ABRs were measured with an evoked potential recording system (NEC, Tokyo). The peak amplitude was measured peak to trough and the threshold was defined as 1 μV.
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0344825017
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Horizontal sections of the temporal bone showed no gross difference between wild-type and mutant mice in the thickness of the stapes footplate or in the size or shape of the bony labyrinth, including the internal auditory meatus.
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0345687848
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Under deep anesthesia, cochleas from 11-week-old mice were fixed with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4] overnight at 4°C. The specimens were decalcified with 0.12 M EDTA, dehydrated, and embedded in paraffin. Serial sections (6 μm) were stained with hematoxylin and eosin. Five sections containing the suprastrial zone of the midbasal turn were selected at random and the number of fibrocytes (mean ± SD) was counted in sections from wild-type (590 ±n 25) (n = 4) and mutant (605 ± 50) (n = 4) male mice.
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0344825016
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Mice at postnatal day 0 were anesthetized and perfused with ice-cold 4% PFA and immersed in 4% PFA at 4°C overnight Heads were embedded in OCT compound (Sakura) and cyosectioned (20 μm). Digoxigenin-labeled RNA probes were synthesized by using a 390-base-pair fragment of the 3′ noncoding region of mouse Brn-4 as a template (Boehringer). Tissue sections were treated with 4% PFA, washed in phosphate-buffered saline, and then hybridized with the probes at 72°C for 24 hours. Sections were washed under stringent conditions and hybridization signals were detected by immunohistochemical analysis.
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B. A. Schulte and J. C. Adams, J. Histochem. Cytochem. 37, 127 (1989); S. S. Spicer and B. A. Schulte, Hear. Res. 56, 53 (1991); T. Kikuchi, R. S. Kimura, D. L. Paul, J. C. Adams, Anat. Embryol. 191, 101 (1995); S. S. Spicer and B. A. Schulte, Hear. Res. 100, 80 (1996).
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Mice were anesthetized and perfused with 2.5% glutaraldehyde in 0.1 M sodium cacodytate buffer (pH 7.4). Cochleas were fixed by perilymphatic perfusion with 2.5% glutaraldehyde overnight at 4°C and immersed in the same fixative for 2 more hours at room temperature. Electron microscopy was performed as in (79).
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0345687846
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In both wild-type and mutant mice, all three cell types of the stria vascularis (marginal, intermediate, and basal cells) were identified and presented a normal appearance. There was no observable difference in the structure of intrastrial capillaries between wild-type and mutant mice. Myelinated axons from spiral ganglion cells to sensory hair cells were well preserved. At the bases of inner hair cells, the fine structure of the afferent nerve terminal synapses appeared to be normal in mutant mice.
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For EP measurement each mouse was artificially ventilated with a respirator through a tracheal cannula after deep anesthesia and muscular relaxation. Rectal temperature was maintained at 37°C and an electrocardiometer was monitored. A glass microelectrode filled with 150 mM KCl was inserted into the scala media of the basal turn through the lateral wall of the cochlea and the output was recorded with a high-impedance dual electrometer (WPI), New Haven, CT.
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Supported by a grant-in-aid for Scientific Research on Priority Areas (O.M., T.N.); grant-in-aid 07457394 (K.I.) from the Ministry of Education, Sports, Science and Culture of Japan; and a grant from the Ministry of Health and Welfare. We thank M. Ogawa, K. Kaga, and T. Murobushi for valuable discussions; K. Jishage, H. Nishino, R. Sato, and K. Yamaguchi for technical support; and T. Morizono, C. M. Hackney, and D. N. Fumess for support
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