Col1a1-driven transgenic markers of osteoblast lineage progression

The modular organization of the type I collagen promoter allows creation of promoter-reporter constructs with preferential activity in different type I collagen-producing tissues that might be useful to mark cells at differen t stages of osteoblastic differentiation. Primary marrow stromal cell (MSC) and mouse calvarial osteoblast (mCOB) cultures were established from trans genic mice harboring different Col1a1 promoter fragments driving chloramphe nicol acetyltransferase (CAT), In these models, Col1a1 messenger RNA (mRNA) and alkaline phosphatase (ALP) are the first markers of differentiation ap pearing soon after the colonies develop. Bone sialoprotein (BSP) is detecte d 2-3 days later, followed by osteocalcin (OC) expression and nodule minera lization, A 3.6 Col1a1 fragment (ColCAT3.6) initiated activity concomitant with ALP staining and type I collagen mRNA expression. In contrast, a 2.3 C ol1a1 fragment (ColCAT2.3) became active coincident with BSP expression. Th e pattern of transgene expression assessed by immunostaining was distinctly different. ColCAT3.6 was expressed within and at the periphery of developi ng nodules whereas the ColCAT2.3 expression was restricted to the different iated nodules, The feasibility of using green fluorescent protein (GFP) as a marker of osteoblast differentiation was evaluated in ROS17/2.8 cells. A 2,3-ltilobase (kb) Col1a1 promoter driving GFP (pOB(4)Col2.3GLP) was stably transfected into the cell line and positive clones were selected. Subcultu res lost and then regained GFP expression that was localized in small clust ers of cells throughout the culture. This suggests that expression from the 2.3-kb Col1A1 fragment is determined by the state of differentiation of th e ROS17/2.8 cells. Col1a1 transgenes should be useful in appreciating the h eterogeneity of a primary or immortalized culture undergoing osteoblastic d ifferentiation.