A targeting vector was designed to insert a nuclear-localized Cre recombinase gene and polyA signal followed by an FRT-flanked PGK-Neo cassette into the initiation codon of the otoferlin (Otof) locus. This construct was electroporated into C57BL/6-derived Bruce4 embryonic stem (ES) cells. Chimeric mice were bred to C57BL/6 mice to establish the colony. Next, mutant mice were bred with FLPe-expressing mice on a C57BL/6 congenic background to remove the FRT-flanked PGK-Neo cassette. The resulting Otof-Cre mice were then bred to C57BL/6J inbred mice for approximately two generations (selecting away from the FLPe transgene).