Mapping Of The Pathogenic Gene And Analysis Of The Keratopathy Characteristics In An ENU-induced Corneal Disease Mouse Model

N-ethyl-N-nitrosourea(ENU)is a highly effective point mutagen that can generate random mutation in the mouse genome.Following an ENU-mutagenesis screen for mutations,a large number of mouse models of human and other animal diseases were recovered for the study of the occurrences,development mechanisms and gene functions.Here,we report a mouse which was identified as a new ENU-induced mutant with a corneal opacity phenotype,and mapped the pathogenic gene.1.A corneal disease mouse obtained by ENU mutagenesis16 adult C57BL/6J(B6)male mice(G0)were injected intraperitoneally with ENU.The male mice were mated with female mice from the same strain when they recovering its reproduction capacity.A total of 12 mutant mice were obtained in 167 G1 generation mice by screening for visible mutation in their progenies.After genetic confirmation test,2 heritable mutant mouse models(corneal opacity and anophthalmia)were identified in the B6 genetic background.However,after mating with C3H/HeJ(C3)mice,no abnormal phenotype was found in the(B6×C3)F1 background of anophthalmia mouse,Therefore,the following studies are focus on the corneal disease mouse with corneal opacity.2.Mapping of the pathogenic gene that causes corneal disease mouseTo map the pathogenic gene,corneal opacity mice in the B6 genetic background were mated with C3 mice to obtain F1 mice,[(B6×C3)F1×B6]N2 mutant mice and[(B6×C3)F1×C3]N2 mutant mice were bred.For initial mapping,we tested genomic DNA from 15 N2 samples with microsatellite markers across the whole genome.The mutation was mapped to chromosome 10,and had no exchange with marker D10Mit233,which was located at 61.58cM(LODs 4.5),the pathogenic gene in corneal opacity mice was preliminary located on chromosome 10.In order to further refine the map position,genomic DNA from 18 N2 mutants was analyzed using microsatellite markers on chromosome 10.The pathogenic gene was mapped to mouse chromosome 10 between makers D10Mit162 and D10Mit233,which were located at 55.73 and 61.58cM,respectively.3.HE staining analysis and immunohistochemical staining analysis of corneal sections for corneal disease mouseHE staining showed that:wild-type mice have clear corneal structure,arranged neatly epithelial cells,smooth basal layer of epithelium,parallel arranged collagenous fibrils and no blood vessels in the stroma and obvious endothelial cells.The epithelium was markedly thinner and corneal neovascularization were also detected in the corneal stroma in corneal opacity mice.We performed staining for the corneal epithelial cells differentiation marker keratin 12(K12),an epithelial cell differentiation marker keratin 14(K14),and a specific epidermal differentiation marker,Keratin 10(K10)by immunohistochemistry in corneal opacity and control mice at 3 W、6 W and 20W.The results showed that:In the control corneas,K12 was present and K10 was not detected in the corneal epithelium,and the expression of K14 was restricted to basal epithelial cells.In the mutant corneas,K12 expression was weak and negative locally in corneal epithelial,K14 expression was enhanced and positive locally in corneal epithelial,K10 expression was positive locally in corneal epithelial,and the expression of PAX6 decreased significantly in all corneal epithelium.Conclusion:In this study,we report a mouse which was identified as a new ENU-induced mutant with a heritable corneal opacity phenotype,and the pathogenic gene in corneal opacity mice was preliminary located on chromosome 10.HE and immunohistochemical analysis were used to study the characteristics of cornea.Our research lays the foundation for the study of human and other animal corneal diseases.