Dlx5 regulation of mouse osteoblast differentiation mediated by avian retrovirus vector.

Aim: To study the effect of Dlx5 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)-Dlx5 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)Dlx5 consistently expressed increased levels of bone differentiation markers - type 1 collagen (Col1a1), 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. Dlx5 overexpression mediated by an RCAS-BP(A) vector stimulates mouse osteoblastic differentiation in Beta-AKE transgenic mice. Dlx5 induces osteoblast differentiation from bones formed either by endochondral or by membranous ossification.