Characteristics Of Corneal Opacity And Its Modified Gene In RAX Nonsense Mutant Mice

Corneal disease is one of the main blinding of the current eye disease,and is the second most blind eye disease comparing with the first cataract disease in China,accounting about 15.4%.At present,the vast majority disease that threatens human health such as diabetes,hypertension,coronary artery disease and schizophrenia,including congenital defects in children such as cleft lip,E cleft and many congenital heart disease are also subject to multi-gene regulation.In this laboratory,a mouse model of corneal opacity phenotype was obtained by ENU chemical mutagenesis.The main gene was identified as RAX and the mouse was named RAX mutant mouse.The main contents of this paper is the cornea characteristics of RAX mutant mouse and its modified gene,mainly divided into the following three aspects:1.RAX nonsense mutations in miceIn this study,taking RAX nonsense mutant mice as our research target,breeding with normal B6 mice,and filtering heterozygous mice with corneal opacity phenotype.Histological analysis of the eyeballs of the corneal opacity showed that the corneal stroma and cornea of the mutant mice were thinning and the corneal cells were vacuolated.And the molecular proteins of mice over 8 weeks were analyzed by immunohistochemical method,the results show that the expression of related protein such as:Cytokeratin 12(K12).Cytokeratin 14(K14),Cytokeratin 13(K13)and Cytokeratin 10(K10)and were changed significantly.2.Positioning modified gene of corneal opacity in miceTo map the mutant gene,corneal opacity B6 mice were mated with DBA(D2)mice to obtain F1 mice.,[(B6×D2)×B6]N2 mutant mice were bred.We found 180 corneal opacity mice from 1782 N2 mice.After linkage between microsatellite markers and mutant gene,the modified gene was linked to microsatellite D1Mit145 with LOD5.97.In order to precisely locate the modified gene,Adding more microsatellite markers on chromosome 1,and the modified gene was finally located within 68.38cM-74.68cM on chromosome 1.3.Identification for modified gene of corneal opacityAccording to the results of locating,we searched the suspected modified gene near locating region in the MGI,and then analyzed the gene function of suspected modified gene.The candidate gene was identified as Pou2f1.Then,the primers were designed and sequenced.The sequence of the mutant mice compared with the wild type B6 mice,D2 mice gene sequences.The primer names are Pou2f1910,Pou2f11029,Pou2f1556,Pou2f1717,Pou2f1703,Pou2fl736,Two-way sequencing results showed that overlapping peaks was not found at the gene coding region in mutant mice,while found single nucleotide polymorphism in the promoter.