The Molecular Mechanism Of An ENU-Induced Mouse Microphthalmia And Anophthalmia Model

ENU is such a mutagen. This synthetic compound was described as the "most potent mutagen in mice". Following an ENU-mutagenesis screen, a large number of mouse mutants with a variety of phenotypes were recovered. We here describe an ENU-treated B6 male mouse, showed a microphthalmo or anophthalmia that varied in phenotypic severity, and some mice showed eyeless in one side even both sides. Our work is divided into three parts based on the mutant mice.1. Generation of Microphthalmia and anophthalmia mice using ENU mutagenesisScreening G1 mice discovered a mouse with Microphthalmia and anophthalmia. The mutant mouse was mated with wild-type B6 mice to get heterozygous mice. After mating the mutant with B6 mice, a percentage of the progeny (23/100) were recorded to have the Microphthalmia phenotype. The backgrounds of B6 heterozygous mice were mated with DBA (D2) mice to obtain Fl mice. The Fl generation mutant mouse back cross B6 mice, [(B6×D2) F1×B6] N2 generation mutant mouse:21/561 of the progenies showed a similar phenotype.2. Mapping and identification of the mutant gene that cause Microphthalmia and anophthalmiaIn order to locate the position of the mutant gene on the chromosome, we outcrossed heterozygotes on the B6 genetic background to D2 to obtain F1 mice and then back cross B6 mice to obtain N2. We tested genomic DNA from 44 D2 samples exhibiting extensive Microphthalmia and anophthalmia with microsatellite markers across the whole genome. We observed no exchange and significant linkages with other chromosomal loci of the markers D18mit124 and D18mit184. The region between D18mit124 and D18mit184 contains the RAX gene and the phenotypes of other RAX mutants resemble that of our mutant. Therefore, the RAX gene was considered a good candidate for the aganglionic megacolon.On examination of the RAX gene, we found a single nucleotide change, a C/T substitution, in nucleotide position 557. Sequence analysis of RAX gene for microphthalmo homozygous revealed a C/T transversion mutation that resulted in an amino acid substitution at position 187 in the protein of an arginine codon (R) by a stop (nonsense) codon.3. RAX protein function impact analysis result from R187X nonsense mutationTo examine whether the R187X mutation has an impaction on RAX protein function, we assessed whether R187X nonsense mutations have some impactions on RAX proteins associated with DNA sequence combining ability. The in vitro mutagenized R187X cDNA as well as the wild-type plasmid vector were introduced into COS-7 cells, Twenty-four hours after transfection, COS-7 cells were fixed with indirect immunofluorescence and PI staining. Cells were examined using a confocal microscope and found normal and mutant RAX proteins both located in the nucleus, so we exclude R187X mutations affect RAX gene to localize properly to the nucleus. After COS-7 cells transfected twenty-four hours, we extracted nucleoprotein and used in electrophoretic mobility shift assays (EMSA), finally we find the R187X mutation causes premature truncation of the RAX protein and removes the ability of the mutant protein to interact with DNA.