acquisition were performed using the Smart EP evoked potential system (Intelligent Hearing Systems, Miami, FL). Histopathology and scanning electron microscopy Anesthetized mice were perfused through the left ventricle of the heart with phosphate-buffered saline (PBS) followed by Bouin’s fixative. For microscopic analysis of cross-sections, inner ears from mutant and control mice were dissected, perfused with Bouin’s fixative, immersed in the same solution for 24 to 48 hours, decalcified with Cal-EX solution for 6 hours, and embedded in paraffin. Sections (4 mm) were cut, mounted on glass slides, and counterstained in hematoxylin/eosin (H&E). Scanning electron microscopy of inner ear organs was performed essentially as described (Furness and Hackney, 1986). Inner ears were dissected out of the skull, fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 3–4 h at 4uC, and then washed three to four times in 0.1 M phosphate buffer. Hair cells of the organ of Corti were exposed by carefully dissecting away the overlying bone and membrane. Mouse cochleas were processed in osmium tetroxide-thiocarbohydrazide (OTOTO), dehydrated with ethanol, and critical-point dried with hexam- ethyldisilazane (Electron Microscopy Sciences, Hatfield, PA). Samples were mounted onto aluminum stubs and sputter-coated to produce a 10–15-nm gold-palladium coat. Samples were examined at 20 kV with a Hitachi 3000N VP scanning electron microscope. Genetic mapping and progeny testing Genomic DNA for genotyping mice was rapidly prepared from tail tips by the hot sodium hydroxide and Tris (Hot SHOT) procedure [67]. A pooled DNA strategy using microsatellite markers [68] was used to initially localize the rda and rda2J mutations to Chr 9. Individual DNA samples from linkage mice were subsequently genotyped for multiple microsatellite markers and SNPs located on Chr 9. PCR was performed in a Bio-Rad PTC-200 Peltier Thermal Cycler. Amplification consisted of an initial denaturation at 97uC for 30 s followed by 40 cycles, each consisting of 94uC for 30 s (denaturation), 60uC for 30 s (annealing), and 72uC for 30 s for the first cycle and then increasing by 1 s for each succeeding cycle (extension). After amplification, the product was incubated for an additional 10 min at 72uC (final extension). PCR products were separated on 2.5% SeaKem gels (Lonza, Rockland, ME) and visualized with ethidium bromide. For progeny testing, each recombinant mouse was mated with an rda/rda mutant mouse, and progeny were tested for hearing by ABR. If any progeny exhibited elevated ABR thresholds, the genotype of the recombinant parent was classified as +/rda; if at least 10 progeny (from two or more litters) were examined but none showed elevated thresholds, the recombinant parent was classified as +/+. Candidate gene sequence analysis High quality genomic DNA for candidate gene sequencing and Southern blot analysis was prepared from mouse spleens by standard phenol/chloroform extraction and ethanol precipitation methods. To sequence cDNA and assess gene expression, total RNA from individual mouse tissues was isolated with Trizol reagent following the protocol of the manufacturer (Life Technologies Corporation, Grand Island, NY). Mouse cDNA samples were synthesized with the SuperScript First-Strand Synthesis System (Life Technologies). PCR for comparative DNA sequence analysis was performed according to the conditions described above for linkage mapping. Elmod1 Mutations Cause Inner Ear Dysfunction Primers for genotyping and amplifying portions of the mouse Elmod1 gene for sequence comparisons were designed using Primer3 (http://primer3.sourceforge.net/) and synthesized by Integrated DNA Technologies (Coralville, IA, USA). Primer sequences for cDNA amplification are given in Table S2, and those for genomic DNA amplification are given in Table S3. PCR products were purified with the QIAquick PCR Purification Kit (Qiagen Inc., Valencia, CA). Primers used for DNA amplification were also used for DNA sequencing on an Applied Biosystems 3700 DNA Sequencer with an optimized Big Dye Terminator Cycle Sequencing method. Southern and northern blot analyses For Southern and northern blot analysis, DNA probes for specific regions of the Elmod1 gene were generated by PCR amplification of genomic DNA or brain-derived cDNA from C57BL/6J mice with the primers listed in Tables S2 and S3. The PCR fragments were separated on agarose gels, extracted and purified with the QIAquick Gel Extraction Kit (Qiagen, Inc., Valencia, CA, USA). Preparation of Southern and northern blots, probe labeling, and hybridization methods followed previously described standard procedures [69]. Commercially prepared northern blots of mouse embryos and multiple adult tissues (Clontech Laboratories, Inc., Mountain View, CA) were also analyzed. For Southern blots, 20 mg of genomic DNA, predigest- ed with a restriction endonuclease, was loaded per lane on an 0.8% agarose gel and electrophoresed in TAE buffer at 2 V/cm for 12 hours. For northern blots, 15 mg of total RNA was loaded per lane on a 0.8% agarose gel with 2.2 M formaldehyde, electrophoresed in 16 MOPS buffer at 3 V/cm for 4 hr. DNA from Southern blots and RNA from northern blots were transferred to positvely charged nylon membranes by vacuum in 106 SSC and UV cross-linked to the membranes. Probes used for blot hybridizations were labeled with 32P by the random primer method. Gene expression analysis by RT-PCR and 59RACE Multiple tissues from adult wildtype mice were screened for the presence of Elmod1 transcripts by RT-PCR using the wex3F and wex7R primers listed in Table S2. Elmod1 transcripts from brain- derived cDNAs of wildtype mice and rda2J mutant mice were further analyzed with the multiple primer pairs listed in Table S2. Total RNA and cDNAs were prepared as described above for candidate gene sequence analysis. For the Rapid Amplification of cDNA Ends (RACE), we used a commercial kit (FirstChoice RACE-Ready cDNA, Life Technol- ogies, Grand Island, NY) and total RNA from mouse brain tissue. This procedure uses RNA ligase mediated RACE to amplify cDNA only from full-length capped mRNA. Reactions were performed according to manufacturer’s specifications with Elmod1 gene-specific primers given in Table S2 (wex1F, Wex2R1, wex2R2). Antibody preparations and western blot analysis The complete protein coding sequence of Elmod1 was cloned into the 2.1 TOPO vector using the TOPO TA Cloning Kit (Life Technologies, Grand Island, NY). The resulting plasmid was sent to Proteintech Group, Inc (Chicago, IL) where it was cloned into the expression vector pGEX4T-1 for protein production. Purified ELMOD1 protein was then injected into rabbits for polyclonal antibody production. High titer antiserum was collected and affinity purified. Western blots were performed as described previously [70]. Whole brains were homogenized in lysis buffer as described. Blots PLoS ONE | www.plosone.org 10 April 2012 | Volume 7 | Issue 4 | e36074