Determining the Structural and Functional Characteristics of Enamelin in Enamel Formation.

Abstract

Enamelin (Enam) is essential for proper dental enamel formation. In Enam null mice the mineralization front associated with the secretory surface of the ameloblast membrane fails to initiate and elongate enamel mineral ribbons, so no true enamel forms. The mineralization front is a complex of enamel proteins that cannot be reconstituted in vitro. The main purpose of this dissertation was to develop an in vivo assay to study the structural and functional characteristics of enamelin in enamel formation by establishing a range of enamelin transgene expression that recovers the enamel phenotype of Enam knockout mice. A mouse expression vector was constructed using 4.6 kb of 5’ amelogenin gene up to the translation initiation codon, the 3.8 kb Enam cDNA, and 1.1 kb of amelogenin 3’ noncoding sequence. This construct was assembled and used to generate enamelin transgenic mice. The expression level of Enam gene in different transgenic mice lines varies from very little to as high as around 5 times as the amount of endogenous gene expression. Using these transgenic mice to breed with Enam knockout mice, we found that appropriate transgene expression level can fully recover the defective phenotype of Enam knockout mice. In one transgenic mouse line, which has a moderate expression level, the transgene successfully rescued the defective phenotype of Enam knockout mice. The enamel thickness and microstructure of enamel is the same as wild-type mouse in this transgene expressed in knockout background mice. Other transgenic mouse lines have a normal enamel thickness and normal decussation pattern when the transgene is expressed in heterozygous background. The teeth appear yellowish and smooth, which is similar to that of the wild-type mouse. We also found in moderate to high expression level enamelin transgenic mouse lines, the enamel started to have defective phenotypes: with moderate expression level, the teeth show protrusive structure on the surface of enamel; with high transgene expression, the enamel layer is almost lost. This is the first time in the literature that described the effect of over-expression of enamelin in developing enamel.