An In Vivo Model to Study Osteogenic Gene Regulation: Targeting an Avian Retroviral Receptor (TVA) to Bone With the Bone Sialoprotein (BSP) Promoter

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Abstract
To study bone development in vivo, a transgenic mouse model was established in which an avian retroviral receptor (TVA) gene driven by the BSP promoter was selectively expressed in skeletal tissues. The model was validated by showing suppressed BSP expression and delayed bone and tooth formation after infection with a virus expressing a mutated Cbfa1/Runx2 gene. Introduction: Tissue‐specific expression of the avian retroviral (TVA) receptor can be used to efficiently target ectopic expression of genes in vivo. To determine the use of this approach for studies of osteogenic differentiation and bone formation at specific developmental stages, transgenic mice expressing the TVA receptor under the control of a 5‐kb bone sialoprotein (BSP) promoter were generated. The mice were first analyzed for tissue‐specific expression of the TVA gene and then, after infection with a viral construct, for the effects of a dominant‐negative form of the Cbfa1/Runx2 transcription factor on bone formation. Materials and Methods: We first generated transgenic mice (BSP/TVA) in which the TVA gene was expressed under the control of a 4.9‐kb mouse BSP promoter. The tissue‐specific expression of the TVA gene was analyzed by RT‐PCR, in situ hybridization, and immunohistochemistry and compared with the expression of the endogenous BSP gene. A 396‐bp fragment of mutated Cbfa1/Runx2 (Cbfa1mu) encoding the DNA‐binding domain was cloned into a RCASBP (A) viral vector, which was used to infect neonatal BSP/TVA mice. Results and Conclusion: Expression of the TVA receptor mRNA and protein in the transgenic mice was consistent with the expression of endogenous BSP. Four days after systemic infection with the Cbfa1mu‐RCASBP (A) vector, RT‐PCR analyses revealed that the expression of BSP mRNA in tibia and mandibles was virtually abolished, whereas a 30% reduction was seen in calvarial bone. After 9 days, BSP expression in the tibia and mandible was reduced by 45% in comparison with control animals infected with an empty RCASBP vector, whereas BSP expression in the membranous bone of calvariae was decreased ∼15%. However, after 4 and 8 weeks, there was almost no change in BSP expression in any of the bone tissues. In comparison, a reduction in osteopontin expression was only observed 9 days after viral transfection in the three bones. Histomorphological examination revealed that bone formation and tooth development were delayed in some of the mice infected with mutated Cbfa1. These studies show that BSP/TVA transgenic mice can be used to target genes to sites of osteogenesis, providing a unique system for studying molecular events associated with bone formation in vivo.