| Background and objectionDeafness is a serious disease which affects the most common human quality of life. The countries all over the world through the epidemiological investigation of congenital deafness in newborns in proportion reached 1/1000-3/1000.Animal models play an important role in the study of genetic deafness. The deaf minipigs that we found can be in accordance with the need to establish animal model of single gene genetic pedigree for verification of genetic rules. The deaf minipigs provides an ideal genetic material for genetic analysis and gene mapping and the discovery of new genes. The Bama miniature pigs have strong reproductive capacity and low feeding cost. Whole genome sequencing of the Bama miniature pigs had been completed. Therefore, the genetic albinism deafness pedigree miniature pigs will be useful to elucidate the mechanism of deafness with good application prospect. The research content of albinism deafness miniature pigs family provide experimental basis for the research and treatment of human hearing mechanism. Based on the construction of the deaf miniature pig family, the related functions reseach, pathological morphology reseach, the gene mapping and molecular biology were studied. There will be good prospects in the research of the mechanism of hearing loss, and the research of cochlear transplantation.Methods1. Family establishment Family establishment Firstly, Bama boar was inject 105mg/kg ENU in The ear vein to find mutagenesis. By sow breeding with mutation sperm, mutant F1 Generated. In the same ways, F2 and F3 were born. After expanding reproduction, dominant mutation pedigree was established.2. Study on the characteristics of disease model2.1 The Smart EP evoked potential instrument, Bama miniature pigs of normal andalbinism deafness were auditory brainstem response, by testing 3 times and takeing the average value.2.2 By paraffin section, celloidin section of normal and albinism deafness Bama miniature pig skin, eyes and the cochlea were examined.2.3 The albinism deafness Bama miniature pigs were detected by scanning electron microscope.2.4 Study of normal and albinism deafness Bama miniature pig cochlea were examined by high resolution spiral CT, MRI magnetic resonance of water imaging.3. Gene mapping By GWAS method of association and linkage analysis, genes were detected in albinism deafness Bama miniature pig.Results1. Family establishment By ENU injection,Bama miniature pigs can produce mutation of sperm. We found that this mutation of sperm can produce a F1 generation with deafness mutations. The deafness mutation can be inherited to the offspring. We successfully established a dominant mutation family with deafness mutation by propagation method.2. Study on the characteristics of disease model2.1 Auditory brainstem response(ABR) test results: by listening testing, we found that the albino Bama miniature pig is severe deafness. The normal and mutant Bama miniature pigs were anesthetized. Brainstem auditory evoked potential Smart EP auditory brainstem response(ABR) test, the results showed that all the parameters measured in the albino deafness Bama miniature pigs in each frequency instrument maximum output of 110 d B cannot lead to ABR peak, which means severe deafness. But the normal Bama miniature pigs had normal hearing, by ABR peak of 40~60d B SPL.2.2 Cochlear celloidin section experiment results: by the cochlear celloidin section, we found that cochlea basal turn of albinism deafness pig is normal, but it is not fully developed for bony labyrinth cochlea and endolymphatic collapse, the atrophy of stria vascularis, hair cells and spiral ganglion cell degeneration. So hearing loss is Mondini deformity. By the paraffin sections of skin and eye detection experiments, we found: Animal iris shallow features, the cornea pupil region than the common Bama miniature pig Brown became pale. Histological observation showed that the mutation of the retinal pigment epithelial cell layer of melanin particles less than RPE cells in normal individuals. By comparison of normal pigs and mutational pig skin whitening deafness, we found: black pigment in albino mutational pig was significantly reduced, compared with the normal pig.2.3 Bama miniature pigs were detected by scanning electron microscope. We found that the structure of cochlea and pig are similar, including the structure, the 3 semicircular canal and vestibular hair cells. Only the cochlear turns in wild type pig is 3.5 rings. There are a total of 4 rows of hair cells with only one row of inner hair cells. Arrayed along the basilar membrane are inner hair cells and outer hair cells.2.4 High resolution spiral CT, MRI magnetic resonance imaging: according to the experimental results, we found the pigcochlea albino deafness for Mondini malformation. The deaf and normal pigs were detected respectively by high resolution spiral CT, MRI magnetic resonance of water imaging. Ultrastructure examination showed normal, Bama miniature pig cochlea for 3 and a half circle, and the mutation of porcine spiral only 1 and a half circle, the circle of the cochlea basal normal the top and second laps, fusion, and no enlargement of vestibular aqueduct and other pathological change, with Mondini malformation.3. Gene mapping By GWAS method of association and linkage analysis, gene of albinism deafness Bama miniature pig was mapped. Genetic linkage analysis found that about more than 100 gene regions malformations of inner ear gene located on chromosome 5, in the region of SOX10 gene was sequenced candidate genes. The Gene mapping results of hereditary deafness Bama miniature pig(TB007T095XXXX): The mutations is in second exon of SOX10 gene(c.325A>T, R109W). The mutation site is located in 109 amino acids. Mutations of deafness and albino trait in pigs were separated, the prevalence of heterozygous for AT, normal pigs were homozygous AA.ConclusionsBama boar was injected N-nitroso-n-ethylure(ENU) in the ear vein to find mutagenesis. By sow breeding with mutation sperm. We found that the deaf pig and established the family of the deaf pig.GWAS method was used to locate the mutagenesis by located at 109 amino acids(AT in inner ear malformation pigs) in the inner ear malformation animal(TB007T095XXXX). By ABR, MRI, CT, paraffin section, collodion gel slice, transcriptome inspection found that the inner ear malformation pigs of pathological and physiological changes.This study showed it was feasible to establish a large mammalian model of hereditary inner ear malformationby mutagenesis method. Gene mapping of mutation of the autosomal dominant inner ear malformation can be done by GWAS method.In this study, Animal model of human deafness for swine laid a solid foundation by construction of a pig family of hereditary inner ear malformation, gene mapping, studying on the characteristics of the disease. About the study of hereditary deafness pig, it will be elucidated in the pathogenesis of deafness, cochlear implant, and so on. It had a good application prospect as genetics disease large animal model. |
PDF Full Text Request