deletion. Founder animals were identified, and heterozygous progeny were delivered to University of California, Berkeley. Heterozygous animals were bred to generate homozygous Clrn1 KO animals. WT C57BL/6J control, Clrn1 heterozygous (+/2), and Clrn1 homozygous (2/2) mice were maintained up to 2 years for histological, histochemical, electrophysiological, and biochem- ical analyses. All experiments were performed in adherence to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and were also approved by the Animals Care and Use Committee of the University of California, Berkeley. Nucleic Acid Isolation and Reverse Transcription Total RNA was isolated from numerous tissue sources, including WT adult C57BL/6J mice, Clrn1 KO (2/2,+/2) mice, C3H/HeJ (C3H) mice, WT Sprague-Dawley (SD) rats, P23H-3 (rhodopsin mutant) transgenic rats, and Royal College of Surgeons (RCS) rats. For adult retinas and brain regions, tissues samples were carefully dissected from any surrounding tissue. For fetal tissues, timed pregnancies were used, and whole fetuses, heads, brains, eyes, or retinas were dissected. Tissues were immediately homogenized in a 1.5 mL microfuge tube containing 100–300 mL of Tri-Reagent (Sigma-Aldrich) using a small plastic sterile pestle (Fisher Scientific, Pittsburgh, PA). Total RNA was extracted, precipitated, and resuspended in ddH2O according to manufacturer’s instructions. For cultured M u¨ller cell studies (a gift from Vijay Sarthy), 6-well or 10 cm plates of cells were grown to near confluence, and total RNA was isolated using an RNeasy kit (Qiagen Corp., Valencia, CA) per the manufacturer’s instructions. Purity was assessed and total RNA was quantified with 260 and 280 nm light. RNA was reverse transcribed using M-MLV reverse transcriptase (Promega) according to manufacturer’s instructions. A standard reverse transcription reaction was performed for 2 hours at 37uC, and included 25–50 ng/mL input total RNA. After the 2 hour transcription reaction, samples were stored at 220uC until used for PCR reactions. Reverse Transcription and Polymerase Chain Reaction All oligonucleotide DNA primers (Figure S1) were ordered from Operon Technologies (Huntsville, AL). Multiple mouse-specific primer pairs were designed to elucidate Clrn1 splice variant expression, developmental expression, and tissue distribution. Total RNA samples were reverse transcribed using M-MLV reverse transcriptase. Twenty-five to 50 ng of cDNA was used as template in all reactions, and all were amplified for 30 cycles, utilizing a 65uC annealing temperature. Polymerase chain reaction (PCR) was performed according to standard thermocycling conditions, using 2.5 mM Mg++, 200 nM primers, 0.1 U Taq Polymerase, 200 nM dNTPs, and 1X PCR buffer. Cycling conditions were typically 60uC for 30’’ (annealing), 72uC for 19 (extension), and 94uC for 20’’ (melting) for 30–35 cycles. Reaction products were electrophoresed through 1–2% agarose gels containing 100 ng/mL ethidium bromide, buffered by Tris- Acetate, pH 8.0, and photographed on a Kodak EDAS 290 imaging system (Fisher Scientific). Real-Time Quantitative RT–PCR Quantitative real-time RT-PCR was used to compare expres- sion of the two primary splice variants, and for quantitative gene expression in the C3H/HeJ (C3H) mouse retina. For splice variant analysis, FAM/BHQ probes (Biosearch Technologies, Novato, CA) were designed to span exon splice junctions (probes crossed the exon 1/3 or exon 1/4 junctions) in order to distinguish between the highly similar transcripts. Expression was normalized to 28S ribosomal RNA for all 6 WT mice. For quantitative gene Clarin 1 Is Necessary for Hearing but Not Vision expression in animal models of retinal degeneration (C3H mice, RCS and P23H-3 rats), SYBR green was the reporter molecule used to assess expression of several retinal transcripts: cFos, Slc1a3, Grm1, Rhod, Rp-1, Thy1, Clrn1, and Vim. Two eyes for each strain/ genotype were assayed, each measured in duplicate, and the average expression levels are shown. Amplification, detection, and quantification were performed on an Mx3000P real-time thermo- cycler (Stratagene Corp., La Jolla, CA). Refer to Figure S1 for primers used. Twenty-five or 50 ng of cDNA was included in each reaction, along with 300 nM primers (final concentration) and 2X Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). The reactions were cycled 45 times with a final melting curve analysis at the end of the experiments to qualify primer specificity. All primer pairs showed single peaks in the melting curve analysis (data not shown). In Situ Hybridization Animals were housed in the Department of Comparative Medicine at the University of Washington and were euthanized according to approved protocols. Timed pregnant female mice were sacrificed and embryos removed at E18.5. The embryos were fixed in a modified Carnoy’s solution (60% ethanol:4% formal- dehyde:10% acetic acid) overnight at 4uC. The samples were washed and dehydrated in 100% ethanol overnight at 4uC, and then were embedded in paraffin and 6 micrometer sections were collected. For the postnatal ages, we noted the day of birth as P0 and sacrificed pups at P0 and P3 according to approved protocols. We then dissected the retinas from the animals, and fixed them and processed them for paraffin sectioning as described above. At least 3 animals were examined at each time point. Mouse Clrn1 cDNA (clone ID: 40130533) to exons 1 and 4, including 154 bp and 646 bp of 59 and 39 UTR sequence was obtained from Open Biosystems Inc. Digoxigenin (DIG)-labeled probes were prepared according to the manufacturer’s manual for DIG-11-UTP (Roche, Indianapolis, IN) and the hybridization was carried out according to Hayashi et al. [68]. The in situ product was visualized using anti-DIG alkaline phosphatase conjugated secondary antibody (Roche) and NBT/BCIP liquid substrate system (Sigma, St. Louis, MO). After ISH, the slides were fixed with 4% paraformaldehyde for 1 hour and washed in PBS. The slides were then incubated with 10% fetal bovine serum and 2% nonfat dry milk in PBS/ 0.1% Triton X-100 (PBST) for 30 minutes. After an overnight incubation with the primary rabbit antibody (anti-Glast, #AB1782, Millipore Corp., Billerica, MA) at 1:200 dilution, or rabbit anti-beta-tubulin III (TuJ1, Covance, Austin, TX) at 1:300 dilution, the slides were washed and incubated in fluorescent conjugated secondary antibody, rinsed with PBST and mounted under a cover glass with Fluoromount G (Southern Biotechnology, Birmingham, AL). Images were captured with a Zeiss Axioplan microscope using a SPOT CCD camera (Diagnostic Instruments, Sterling Heights, MI) and similarly processed using Adobe Photoshop (Adobe Systems, San Jose, CA). Laser Capture Microdissection LCM was used to isolate total RNA from the three nuclear layers of the retina: outer nuclear layer (ONL), INL, and ganglion cell layer (GCL). Briefly, whole eyes were enucleated and immediately frozen in tissue freezing medium in a dry ice/ethanol slurry. Seven mm retinal sections were collected onto ProbeON Plus microscope slides (Fisher Scientific), and stored in 70% ethanol at -25uC. Slides were dehydrated at RT in graded ethanol series (80%, 90%, 36100%) and 2 changes of 100% xylenes, then air dried in a fume hood, and immediately processed with a PixCell II laser capture system (Arcturus, Mountain View, CA). PLoS Genetics | www.plosgenetics.org 13 August 2009 | Volume 5 | Issue 8 | e1000607