Usher syndrome is the most common cause of inherited deafness found in combination with blindness. All Usher patients suffer progressive retinitis pigmentosa, with the degree of hearing impairment and the presence or absence of vestibular function differing among subtypes. A cryptic splice site mutation (216G-->A) in exon 3 of the USH1C gene on chromosome 11p, which encodes a PDZ-domain protein, harmonin, was found in Acadian Usher type IC patients in south Louisiana. In vitro analysis using constructs containing the mutant 216A and subsequent analysis of patient cell lines revealed a deletion of 35 bases in the transcript. In order to analyze the impact of this frame-shift mutation, we created a knock-in mouse model containing the human 216G-->A mutation. A targeting construct was made containing 5' and 3' homology arms, each 4kb in length, and a 650 base pair fragment containing exons 3 and 4 of human USH1C cloned from an Acadian patient homozygous for the 216A mutation. W4/129S6 embryonic stem (ES) cells were electroporated with the targeting construct, and after 10 days of neomycin selection, clones were picked and screened by polymerase chain reaction (PCR) and Southern blot analysis for homologous recombination. Two positive clones for targeted insertion were microinjected into C57BL/6 blastocysts which were then transplanted into pseudo-pregnant females. Chimeras were bred with Cre recombinase-expressing mice for simultaneous deletion of the neomycin gene and germline transmission of the 216A allele. Homozygous Ush1c216A (216AA) mice are hyperactive, display circling and head tossing behavior, and do not have a Preyer reflex at 21-25 days old. RT-PCR analysis of the cochlea and retina from 216AA mice shows the same 35 base deletion characteristic of Usher IC patients.