The Molecular Mechanism Of An ENU-induced Corneal Opacity Mouse Model

N-ethyl-N-nitrosourea (ENU) is a powerful point mutagen that can generate random mutations in the genome. Following an ENU-mutagenesis, a large number of mouse mutants with a variety of phenotypes were recovered for the study of gene function and the generation of human disease models. Here, we report a mouse was identified as a new ENU-induced mutant with a corneal opacity phenotype, and found the loci of mutant gene.1. A corneal opacity phenotype obtained by ENU mutagenesisENU was intraperitoneally injected in forty C57BL/6J (B6) male mice, the male mice were mated with the same strain female mice when they have fertility, their progenies were screened for visible mutation. We found a large number of mutation including polydactyly, microphthalmia, shortail, dilated pupil, corneal opacity. The founder corneal opacity male mouse, which was the progeny of an ENU-treated B6 male mouse and an untreated B6 female mouse, had a corneal opacity phenotype. After mating the mutant with B6 mice, a percentage of the progeny (15/63) were recorded to have the corneal opacity phenotype. Histological analysis revealed stromal neovascularization and obvious proliferation of fibroblast, diffuse staining of the corneal surface using fluorescein reveals a breach in barrier function in the mice compared with wild-type mice.2.Mapping of the mutation gene that causes corneal opacity phenotype of mouseTo map the mutant gene, corneal opacity B6 mice were mated with DBA(D2) mice to obtain F1 mice., [(B6×D2)F1 ×B6] N2 mutant mice were bred. We found 26 corneal opacity mice from 612 N2 mice. Microsatellite markers distributed equally on the mouse chromosome were used to scan the genome of these 26 N2 mice. After linkage between microsatellite markers and mutant gene, the mutant gene was linked to D12Mit285 with LOD3.78. Adding more microsatellite markers and mutant gene was located within 9.04cM on chromosome 12.3. The sequence analysis of gene that causes corneal opacity phenotype of mouseSearching and analyzing gene within 9.04cM on chromosome 12,we found 4 genes linked with eye development:mycn、adam17、sdc1、osr1. Pairs of primers were designed to aim for 4 genes, these genes is amplified by PCR and RT-PCR, and products were recycled and sequenced, then compared with normal B6 mice. Results showed that mycn coding region of 1217th bp in normal B6 mice is T as well as C in corneal opacity mouse, Amino acid sequence prediction indicates that the mutation causes the 406th valine (V) of MYCN protein is replaced by alanine (A), the mutation occurs in the basic helix-loop-helix (bHLH) domain of MYCN protein. Conclusions. The study obtained a corneal opacity mouse, provided materials for researching related diseases.The study first discovered mutation of mycn can cause corneal opacity phenotype in mice, but its mechanism is unknow, further investigate will rich the biology function of mycn gene.