Generation A Jag1 Gene Mutation Eye Dysmorphology Mouse Model Using ENU Mutagenesis

N-ethyl-N-nitrosourea(ENU)is a powerful point mutagen that can generate random mutation in the mouse genome.Following an ENU-mutagenesis screen for mutations,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 which was identified as a new ENU-induced mutant with a heritable eye dysmorphology phenotype,and found the loci of mutant gene1.A mouse with eye dysmorphology phenotype obtained by ENU mutagenesis and its histological analysis.20 adult C57BL/6J(B6)male mice(GO)were injected intraperitoneally with ENU.The male mice were mated with female mice from the same strain when they recovering its reproduction capacity,then,we screened for visible mutation in their progenies.After genetic confirmation test,4 heritable mutant mouse models have been identified,and the following study are focus on one of the mutant mouse with iridocoloboma phenotype.After mating the mutant with B6 mice,a percentage of the progeny(56/120)were recorded to have the corneal opacity or iridocoloboma(or both).HE examination of eye sections from eye corneal opacity mice revealed vacuoles in the surface of the corneal epithelial cells,collagen fibrils were loose in the corneal stromal layer and some endothelial cells were absent.We performed staining for the corneal epithelial cells differentiation marker keratin 12(K12),the epithelial cell differentiation marker keratin 14(K14),and a specific epidermal differentiation marker,keratin 10(K10),by immunohistochemistry in corneal opacity and control littermate mice at 4W and 8W.Immunohistochemistry showed that:(1)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.(2)In the mutant corneas,there was no significant change in K12 expression in corneal epithelium at 4W.But at 8W,K12 expression in the vacuole regions of corneal epithelium was decreased.K14 was highly expressed at 4W and 8W.K10 was not detected2.Mapping of the mutation gene that causes eye dysmorphology phenotype of mouseTo map the mutant gene,eye dysmorphology mice on the B6 genetic background were mated with C3He/J(C3)mice to obtain F1 mice,[(B6×C3)F1×B6]N2 mutant mice were bred For initial mapping,we tested genomic DNA from 16 N2 samples with microsatellite markers across the whole genome.The mutation was mapped to chromosome 2,and had 1 exchange with marker D2Mit249,which was located at 54.07cM(LODs 3.19).In order to further refine the map position,genomic DNA from 122 N2 mutants was analyzed using microsatellite markers on chromosome 2.The mutation was mapped to mouse chromosome 2 between makers D2Mit107 and D2Mit423,which were located at 65.13 and 73.57cM,respectively.3.Sequence analysis of the mutant gene that causes eye dysmorphology phenotype of mouseAfter searching and analyzing genes between 65.13cM and 73.57cM on chromosome 2,the Jag1 gene was finally selected as a candidate gene for this mutation.The exons and flanking intronic sequences of Jagl were amplified from mutant and B6 RNA using RT-PCR and genomic DNA using PCR.It was found that:starting from exon 25,continuous overlapping signals appeared in the sequencing of the mutant heterozygous RT-RCR product,suggesting abnormal mRNA splicing.The RT-PCR product was T-A cloned and sequenced again,and the flanking sequence of exon 25 was then sequenced.It was found that the AG base at the 3' end of the intron 24 in the Jag1 gene on one chromosome of the mutant heterozygous mouse was replaced by the GG base,resulting in the deletion of 7 bases in exon 25.Translation of the mutant allele would produce frameshift and truncated protein.Here,we report a mouse was identified as a new ENU-induced mutant with a heritable eye dysmorphology phenotype,and found the loci of mutant gene.It was found that the mutation phenotype of this model mouse was caused by a new allelic mutation of Jag1 gene.Although it has been reported that Jagl gene mutation can cause corneal abnormalities in mice,there is no in-depth study of corneal histological lesions.The mouse model of Jag1 gene mutant eye dysmorphology established in this experiment provided a new model for the related research of human eye dysmorphology diseases.