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


METHODS
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.


CONCLUSIONS
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.
Erceg
Ivana
I
Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, USA. ivana.erceg@st.hinet.hr
Tadić
Tade
T
Kronenberg
Mark S
MS
Marijanović
Inga
I
Lichtler
Alexander C
AC
D000818:Animals; D015152:Blotting, Northern; D001854:Bone Marrow Cells / Q000502:physiology; D002454:Cell Differentiation / Q000235:genetics*; D002478:Cells, Cultured; D018507:Gene Expression Regulation, Developmental; D005822:Genetic Vectors; D018398:Homeodomain Proteins / Q000235:genetics*; D017403:In Situ Hybridization; D051379:Mice; D008822:Mice, Transgenic; D010006:Osteoblasts / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D012190:Retroviridae / Q000235:genetics*; D012680:Sensitivity and Specificity
C501120:Dlx5 protein, mouse; D018398:Homeodomain Proteins; D012333:RNA, Messenger