Dlx5 regulation of mouse osteoblast differentiation mediated by avian retrovirus vector

Aim. To study the effect of DIx5 introduced by replication-competent avian splice-acceptor (RCAS) in mouse calvarial and bone marrow stromal cells, and to demonstrate that RCAS vector can be a useful system for studying gene expression in mammalian cells derived from beta-AKE mouse. Method. beta-AKE mouse used in experiments is a transgenic mouse line expressing the receptor for the Bryant polymerase subgroup A of RCAS vector (RCAS-BP(A) vector). Primary calvarial osteoblast cultures were obtained from 7-day-old beta-AKE mice. Bone marrow stromal cells were derived from the long bones of 8-week-old beta-AKE mice. Expression of genes cloned into RCAS vector in mouse cells was first established by detecting green fluorescent protein (GFP) in cells infected with RCAS-BP(A)-GFP sapphire by using fluorescence microscopy. Cells were then infected with RCAS-BP(A)-DIxD or RCAS-BP(A) alone as a control, for three days. After differentiation, cells were harvested for mRNA analysis at different time points (day 6 or 7, 11 or 12, 14 or 18, and 21 or 25). The cells were cultured in the presence of ascorbic acid and beta-glycerophosphate, which promotes osteoblastic differentiation. Results. Mouse calvarial and bone marrow stromal cells infected with RCAS-BP(A)-GFP sapphire were fluorescent compared with the controls. Both types of cells infected with RCAS-BP(A)DIx5 consistently expressed increased levels of bone differentiation markers - type 1 collagen (Col1alpha1), osteocalcin, and bone sialoprotein mRNA. Conclusion. RCAS-BP(A) vector transduction of cells from beta-AKE mice is a useful system for studying the role of gene expression in mouse osteoblastic cells. DIx5 overexpression mediated by an RCAS-BP(A) vector stimulates mouse osteoblastic differentiation in beta-AKE transgenic mice. DIx5 induces osteoblast differentiation from bones formed either by endochondral or by membranous ossification.