Null Mutation Of The Fascin2 Gene By TALEN Leading To Progressive Hearing Loss And Retinal Degeneration In C57BL/6J Mice

Objective:Fascin2(Fscn2)is an actin cross-linking protein that is mainly localized in retinas and in the stereocilia of hair cells.Recent studies have indicated that a missense mutation in mouse Fascin2(Fscn2)gene(Fascin2 p.R109H)can contribute to the early onset of hearing loss in DBA/2J mice.To explore the function of the gene,Fscn2 was knocked out using TALEN(transcription activator-like effector nucleases)under the background of C57BL/6J mice.Four mouse strains with deletions of 1,4,5,and 41 nucleotides in the target region of Fscn2 were developed.F1 heterozygous(Fscn2+/-)mice carrying the same deletion of 41 nucleotides were mated to generate the Fscn2-/-mice.Methods:The wild-type mice(Fscn2+/+),heterozygous mice(Fscn2+/-),and homozygous mice(Fscn2-/-)were genotyped by using polymerase chain reaction(PCR).Transcription of Fscn2 was evaluated bu using RT-PCR.Fscn2 expression was detected by using Western blotting.Immunolocalization observation of Fscn2 in retinas and cochleae.ABR thresholds and DPOAE amplitudes were measured in mice in a time-course manner(from 3 to 52 weeks).Degeneration of hair cells(stained for F-actin with Alexa Fluor-488-labeled phalloidin)in the cochleae of mice were observed at various ages(from 3 to 52 weeks).Meanwhile,alterations of the stereocilia in the cochlear basal turns was observed by using scanning electron microscopy(SEM).Dark-adapted(scotopic)flash electroretinograms(ERGs)were conducted of mice at ages 4,8,12,and 24 weeks.At the same time,morphological changes of retina was observed.Results:The Fscn2-/-mice showed progressive hearing loss,as measured in the elevation of ABR thresholds and amplitudes decline of DPOAE.The hearing impairment began at age 3 weeks at high-stimulus frequencies and became the most severe at age 24 weeks.Moreover,degeneration of hair cells and loss of stereocilia were remarkable in Fscn2-/-mice,as revealed by phalloidin staining and SEM.Furthermore,comparing to the controls,the Fscn2-/-mice displayed significantly lower ERG amplitudes and thinner retinas at 8,16,and 24 weeks.Conclusion:A null mutation of Fscn2 in C57BL/6J mice leads to progressive hearing loss and retinal degeneration.In the background of C57BL/6J mice,the null mutation mouse model of Fscnl gene was successfully prepared by using TALEN technique.This model will aid in functional studies of Fscn2 to increase understanding of the etiology of FSCN2-associated disorders in humans.