Functional studies of Osteoadherin by tissue-specific over-expression in mice

Objectives: we sought to investigate functional properties of the small leucine-rich proteoglycan Osteoadherin (OSAD), a protein with restricted expression to bone and teeth. Since knock-out experiments have failed to show any associated phenotype in animals, our approach was to generate a transgenic mouse strain with tissue-specific expression of OSAD. Methods: an expression plasmid with OSAD cDNA under the control of the Osteocalcin promoter was generated and transgenic mouse lines were obtained by using the pronuclear injection technique. Positive transgenic animals were evaluated by gDNA PCR analysis with insert-specific primers and by western blotting. Bone density measurements of tibiae were performed by peripheral Quantitative Computed Tomography (pQCT) on tibiae from male animals at an age of 4 and 16 weeks. Mandibles were sectioned and stained with hematoxylin and eosin. Results: we selected for the clone with the strongest transgenic signal by PCR screening, and western blotting for the construct specific c-Myc tag confirmed expression of the transgenic protein. Heterozygous mating produced a tg/wt offspring distribution according to Mendelian law. Tibiae and mandible ex vivo analysis revealed distinct phenotypical differences in bone and teeth mineral content and quality. Conclusion: we have managed to establish a transgenic mouse strain that over-expresses OSAD in a tissue-specific manner. The Mendelian distribution of offspring shows that the animals are fully fertile and that survival is not dependent on the expression of OSAD. Bone and tooth tissue analysis show a distinct phenotype, which however seems to diminish as the mice age. A possible mechanism could be that OSAD affects the cellular component of mineralized tissues in their ability to deposit and maintain hydroxyapatite or collagen.