Establishment And Study Of L236P Mutation Mouse Model Of Slc26a4 Gene

The protein encoded by the SLC26A4 gene is pendrin,which is expressed in the inner ear and is mainly expressed in marginal cells that can secrete anions.It is an anion transporter and is used to maintain the endolymphatic potential and ion concentration.Mutations in this gene often lead to Pendred syndrome and vestibular aqueduct syndrome in patients,mainly manifested as a sensorineural hearing loss with inner ear malformation,and some will appear dizziness.The degree of deafness varies from person to person,and some are more serious and some are minor.In order to explore the pathogenic mechanism of the gene,there have been many reports on the gene..Previous studies have reported that in the Slc26a4 knockout mouse model,the mice have a complete loss of hearing and most mice have severe balance disorders.These phenotypes cannot mimic the more subtle auditory and balanced phenotypes in human patients.Therefore,the purpose of this project is to create a mouse model that can fully simulate human diseases.Through the analysis of mouse models,the pathogenic mechanism of the gene is explored,and reliable experimental data are provided for clinical practice.In our study,we successfully generated the L236P model of Slc26a4 knock-in mice using the CRESPR/Cas9 technique,which is very common in European and American countries and often detected in the clinical screening of Pendrin syndrome.In our L236P mice,the auditory and balance functions of the mice were detected by ABR and roller experiments,respectively.As a result,it was found that L236P mice have different degrees of hearing loss and balance disorders,which are similar to human phenotypes in patients with the gene mutation.In the auditory aspect,the relatively slight proportion accounted for 21.9%,very serious accounted for 37.5%;in terms of balance,30%of the mice have obvious balance disorders and showed tilting or circling,while others looked normal from the outside.This is similar to the human patient phenotype.Hematoxylin-eosin staining,basement membrane spreading,and scanning electron microscopy,as well as immunofluorescence,were performed on the inner ear of L236P mice which hearing and balance functions were severely impaired.We found that the hair cells were normal in those mice which were 7 postnatal day.But they began to drop slightly at 15 postnatal days,and the hair cells were almost completely lost by one month.By observing the vestibule utricles of L236P mice with severely balance disorders,we found that the otoconia and otoconial membrane were significantly abnormal.The otoconial was no longer located on the otoconial membrane and the otoconial membrane was clearly broken.The stereocilia of the sensory hair cells below the otoconial membrane are severely lost.Therefore,the homozygous knock-in mice L236P of the Slc26a4 gene causes abnormalities in the inner ear cochlear hair cells and vestibular organs of mice,eventually leading to auditory and balance dysfunction.We observer that the phenotypes of L236P mice are fluctuate significantly including different degrees of obstacles.In order to find out the reason,we detected the mRNA expression of Slc26a6,Slc26a11,Slc4a1,Slc4a2,and Slc4a3 related genes of Slc26a4 gene by using fluorescence quantitative PCR assay.As a result,it was found that the mRNA expression level of Slc26a11 gene was significantly up-regulated,while other genes were not changed.Therefore,we hypothesized that the up-regulated of Slc26a11 gene is reason why a part of mice have relatively mild auditory and balance dysfunction.The increase in the amount compensates for the loss of the Slc26a4 gene,which in turn reduces the hearing and balance disorders in mice.