Lastly, while the mutant mouse in the current study and the hearing loss described in patients with DFNA25 are both due to mutations in the gene coding for VGLUT3,

the comparison may not be straightforward. First, it is not certain that the missense mutation described in SLC17A8 is the cause of the hearing loss seen in DFNA25, though a strong correlation was observed (Ruel et al., 2008). Second, the null mutation studied in these experiments would represent a much more severe phenotype than the missense mutation described as potentially causative for DNFA25. Thus, whether this technique could ultimately be beneficial to patients with DFNA25 remains unclear. Despite these differences, as our study documents restoration of normal ABR levels in such a null mutant model, it nonetheless represents an important initial step for the potential treatment PD0332991 manufacturer of inherited deafness. VGLUT3 null mutant mice were generated as described in a C57 (Seal et al., 2008) strain Ponatinib chemical structure then backcrossed with FVB mice (less than seven generations) to obtain a homogeneous genetic background. P1–P12 mice were used for AAV1-VGLUT3 delivery. All procedures and animal handling complied with NIH ethics guidelines

and approved protocol requirements at the University of California, San Francisco (IACUC). All surgical procedures were done in a clean, dedicated space. Instruments were thoroughly cleaned with 70% ETOH and autoclaved prior surgery. Surgery was carried out under a Leica MZ95 dissecting scope and animals were situated with neck extended over solid support. Mice were anesthetized by intraperitoneal injection of a mixture of aminophylline ketamine hydrochloride (Ketaset, 100 mg/kg), xylazine hydrochloride (Xyla-ject, 10 mg/kg), and acepromazine (2 mg/kg) and boosted with one-fifth the original dose

as required. Depth of anesthesia was continuously checked by deep tissue response to toe pinch. Body temperature was maintained with a heating pad and monitored with a rectal probe throughout procedures. Preoperatively and every 24 hr postoperatively, animals were given subcutaneous carprofen analgesia (2 mg/kg) to manage inflammation and pain. Animals were closely monitored for signs of distress and abnormal weight loss postoperatively. Mouse VGLUT3 cDNA was subcloned into the multiple cloning site of vector AM/CBA-WPRE-BGH (kindly provided by R. Palmiter). Human embryonic kidney 293 cells were cotransfected with three plasmids—AAV-mVGLUT3 plasmid, appropriate helper plasmid-encoding rep and AAV1 cap genes, and adenoviral helper pF Δ6—using standard CaPO4 transfection. Cells were harvested 60 hr after transfection, cell pellets were lysed with sodium deoxycholate, and AAV vectors were purified from the cell lysate by ultracentrifugation through an iodixanol density gradient, then concentrated and dialyzed against phosphate-buffered saline (PBS), as previously described ( Cao et al.