Dominant mutations of transmembrane channel-like gene 1 (mice on a C3HeB/FeJ

Dominant mutations of transmembrane channel-like gene 1 (mice on a C3HeB/FeJ strain background have selective degeneration of inner hair cells while outer hair cells remain structurally and functionally intact. severe to profound SNHL, Azacitidine distributor whereas Azacitidine distributor autosomal dominant alleles typically cause postlingual progressive SNHL (Griffith and Friedman 2002). The specific causes of SNHL associated with advanced age (presbycusis) are unknown, but thought to comprise a complex combination of genetic and environmental factors (Schultz 2005). Mutant mice are important tools for identifying these factors, their function in the auditory system, and the pathogenesis of hearing loss (Haider 2002). For example, studies of polygenic age-related hearing loss in inbred mouse strains (Noben-Trauth 2003) facilitated the recent identification of a genetic modifier Azacitidine distributor of hearing loss in humans (Schultz 2005), demonstrating the applicability of mouse models to the dissection of complex hearing loss traits in humans. Dominant and recessive mutations of transmembrane channel-like gene 1 (2002, 2003). encodes a polytopic transmembrane protein of unknown function that is expressed in cochlear hair cells (Kurima 2002; Vreugde 2002). In mice, dominant and recessive mutations of cause SNHL in the Beethoven (2002; Vreugde 2002). Whereas DFNB7/B11 and homozygotes have severe to profound congenital hearing loss, heterozygous service providers of and DFNA36 mutations have delayed-onset, progressive SNHL. SNHL in Beethoven and deafness mice is usually associated with quick degeneration of cochlear hair cells (Bock and Steel 1983; Vreugde 2002), indicating that is required for normal hair cell function or survival. The mammalian cochlea contains two types of hair cells distinguished by their location, morphology, and function (Frolenkov 2004). In general terms, inner hair cells (IHCs) have primarily afferent innervation and function as sensory cells transducing and transmitting auditory signals to the central nervous system. Outer hair cells (OHCs) receive principally efferent innervation and have a distinctive electromotile house postulated to underlie active biomechanical amplification of auditory stimuli. As a result of this active amplification, OHCs generate sounds known as otoacoustic emissions (OAEs) that can be measured noninvasively in living humans and mice with a sensitive microphone in the external auditory canal. In contrast, evaluation of hearing levels by auditory brainstem response (ABR) threshold analysis is an overall measure of peripheral auditory function, including both inner and outer hair cell function. Primary damage or secondary degeneration of OHCs characterizes many common hearing loss phenotypes, such as presbycusis, in humans. Since genetic modifiers are excellent candidates for etiologic determinants of these complex characteristics (Friedman 2000; Schultz 2005), including presbycusis, we sought to identify modifiers of hair cell degeneration in Mouse monoclonal to Dynamin-2 Beethoven mice. MATERIALS AND METHODS Experimental animals: All protocols were approved by the National Institute of Neurological Disorders and Stroke/National Institute on Deafness and Other Communication Disorders Animal Care and Use Committee. Isogenic heterozygous Beethoven (2002) were intercrossed to generate homozygous (C3H-homozygotes were crossed with wild-type C57BL/6J or DBA/2J mice to generate (C/B)F1-and (C/D)F1-hybrids, respectively. F1 hybrids were backcrossed to wild-type parental strains to generate [(C/B)F1 C]N2-and [(C/D)F1 C]N2-Beethoven progeny. genotype analysis: Genomic DNA was prepared from tail clip biopsies by a phenol/chloroform extraction process. A 213-bp fragment of exon 13 was amplified with forward (5-TAT TAA AGGGAC CGC TCT GAA AAC-3) and reverse (5-ATC CAT CAA GGC GAG AAT GAA TAC-3) primers Azacitidine distributor in a 20-l volume made up of 50 ng DNA, 20 pmol of each primer, 200 mol/liter of each dNTP, 1.5 mmol/liter MgCl2, and 1.6 units DNA polymerase. Amplification conditions comprised an initial 2-min denaturation at 95, followed by 30 step-cycles of 30 sec at 95, 30 sec at 57, and 45 sec at 72, with a final elongation of 5 min at 72. PCR products were directly sequenced on an automated sequencer (ABI-PRISM, model 3700; Applied Biosystems, Foster City, CA). ABR and distortion product otoacoustic emissions analyses: ABR thresholds were measured as explained (Szymko-Bennett Azacitidine distributor 2003) with some modifications: we used alternating polarity click and tone-burst stimuli of 47-sec and 5-msec duration, respectively. The number of stimulus presentations was varied from 128 to 1024 depending on signal-to-noise ratio, and suprathreshold stimulus intensities were initially decreased in 10-dB actions followed by 5-dB actions at lower intensities to determine the response threshold. When no waveform was detectable at the highest stimulus level of 90 dB sound pressure level (SPL), the threshold was considered to be 95 dB.