Enamel Structure Defects in Missense Mutation Knock-in Mice.

The () is reported to take part in tooth formation in humans, but the dental phenotype of mutant mice has not been understood. Additionally, the role of the gene in dental hard tissue development is rarely known. In this study, we constructed a missense mutation knock-in mouse model through CRISPR/Cas9 gene-editing technology. Enamel samples from wildtypes (WT) and homozygous mutants (HO) were examined using micro-computed tomography (micro-CT), scanning electron microscopy (SEM), an atomic force microscope (AFM) and Raman microspectroscopy. The results showed that a novel missense mutation (c. 908G>C, p.R303P) knock-in mice model was constructed successfully. The enamel of HO mice incisors appeared chalky and defective, exposing the rough interior of the inner enamel and dentin. Micro-CT showed that HO mice had lower volume and mineral density in their tooth enamel. In addition, declined thickness was found in the unerupted enamel layer of incisors in the HO mice. Using SEM and AFM, it was found that enamel prisms in HO mice enamel were abnormally and variously shaped with loose decussating crystal arrangement, meanwhile the enamel rods were partially fused and collapsed, accompanied by large gaps. Furthermore, misshapen nanofibrous apatites were disorderly combined with each other. Raman microspectroscopy revealed a compromised degree of order within the crystals in the enamel after the mutation. To conclude, we identified enamel structure defects in the missense mutation knock-in mice, which displayed fragmentary appearance, abnormally shaped prism structure, decreased mineral density, altered crystal ordering degree and chemical composition of the enamel layer. This may support the potential role of the gene in the natural development of enamel.