Dacic S, Kalajzic I, Visnjic D, Lichtler A C, Rowe D W
Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington 06030, USA.
J Bone Miner Res. 2001 Jul;16(7):1228-36. doi: 10.1359/jbmr.2001.16.7.1228.
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 different stages of osteoblastic differentiation. Primary marrow stromal cell (MSC) and mouse calvarial osteoblast (mCOB) cultures were established from transgenic mice harboring different Col1a1 promoter fragments driving chloramphenicol acetyltransferase (CAT). In these models, Col1a1 messenger RNA (mRNA) and alkaline phosphatase (ALP) are the first markers of differentiation appearing soon after the colonies develop. Bone sialoprotein (BSP) is detected 2-3 days later, followed by osteocalcin (OC) expression and nodule mineralization. A 3.6 Col1a1 fragment (ColCAT3.6) initiated activity concomitant with ALP staining and type I collagen mRNA expression. In contrast, a 2.3 Col1a1 fragment (ColCAT2.3) became active coincident with BSP expression. The pattern of transgene expression assessed by immunostaining was distinctly different. ColCAT3.6 was expressed within and at the periphery of developing nodules whereas the ColCAT2.3 expression was restricted to the differentiated 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-kilobase (kb) Col1a1 promoter driving GFP (pOB4Col2.3GLP) was stably transfected into the cell line and positive clones were selected. Subcultures lost and then regained GFP expression that was localized in small clusters of cells throughout the culture. This suggests that expression from the 2.3-kb Col1A1 fragment is determined by the state of differentiation of the ROS17/2.8 cells. Col1a1 transgenes should be useful in appreciating the heterogeneity of a primary or immortalized culture undergoing osteoblastic differentiation.
I型胶原蛋白启动子的模块化组织使得能够构建启动子-报告基因构建体，这些构建体在不同的I型胶原蛋白产生组织中具有优先活性，这可能有助于标记成骨细胞分化不同阶段的细胞。从携带驱动氯霉素乙酰转移酶（CAT）的不同Col1a1启动子片段的转基因小鼠中建立了原代骨髓基质细胞（MSC）和小鼠颅骨成骨细胞（mCOB）培养物。在这些模型中，Col1a1信使核糖核酸（mRNA）和碱性磷酸酶（ALP）是菌落形成后不久出现的最早分化标志物。2-3天后检测到骨唾液蛋白（BSP），随后是骨钙素（OC）表达和结节矿化。一个3.6 Col1a1片段（ColCAT3.6）启动的活性与ALP染色和I型胶原蛋白mRNA表达同时出现。相比之下，一个2.3 Col1a1片段（ColCAT2.3）在BSP表达时开始活跃。通过免疫染色评估的转基因表达模式明显不同。ColCAT3.6在发育中的结节内部和周边表达，而ColCAT2.3的表达仅限于分化的结节。在ROS17/2.8细胞中评估了使用绿色荧光蛋白（GFP）作为成骨细胞分化标志物的可行性。一个驱动GFP的2.3千碱基（kb）Col1a1启动子（pOB4Col2.3GLP）被稳定转染到细胞系中并选择阳性克隆。传代培养物失去然后重新获得GFP表达，其定位于整个培养物中的小细胞簇中。这表明2.3-kb Col1A1片段的表达由ROS17/2.8细胞的分化状态决定。Col1a1转基因在认识经历成骨细胞分化的原代或永生化培养物的异质性方面应该是有用的。
