An assay for the determination of biologically active bone morphogenetic proteins using cells transfected with an inhibitor of differentiation promoter-luciferase construct
An assay for the determination of biologically active bone morphogenetic proteins using cells transfected with an inhibitor of differentiation promoter-luciferase construct
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DOI：
10.1016/j.ab.2005.10.030
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发表时间：
2006-02-01
影响因子：
2.9
通讯作者：
Petite, H
中科院分区：
文献类型：
--
作者：
Logeart-Avramoglou, D;Bourguignon, M;Petite, H
骨形态发生蛋白（BMPs）通过调控基因表达，特别是分化抑制因子（Id）基因的表达来控制细胞的命运。这种性质已被利用来创建用于定量活性BMP的高灵敏度测定。用含有与萤火虫荧光素酶报告基因融合的BMP响应元件的表达构建体（BRE-Luc）稳定转染胚胎小鼠细胞（C3 H10 T1/2）。BRE由来自小鼠Id 1启动子的不同序列元件的多聚化产生[15]。向转染子中加入BMP 2（0.5-100 ng/ml）导致细胞裂解物中荧光素酶活性的剂量依赖性增加。这种新的测定法比经典的碱性磷酸酶（ALP）活性测定法灵敏100倍（分别为0.5-1对50- 100 ng/ml），并且更快速（分别为24小时对3-6天，BM P处理）。这种新的测定法对BMP（BMP-2、BMP-4和BMP 7）是特异性的，如其对TGF β 1、bFGF和VEGF的相对不敏感性所证明的。由于其BMP特异性，这种快速，灵敏，非放射性，易于进行的测定可用于监测BMP的生物活性，并最终作为一种基于细胞的筛选测定，以确定和评估调节细胞中BMP信号的分子。(c)2005年爱思唯尔公司All rights reserved.
Bone morphogenetic proteins (BMPs) control cell fate by regulating gene expression, especially inhibitor of differentiation (Id) genes. This property has been exploited to create a highly sensitive assay for quantification of active BMP. Embryonic mouse cells (C3H10T1/2) were stably transfected with an expression construct (BRE-Luc) containing a BMP-responsive element fused to the firefly luciferase reporter gene. BRE results from a multimerization of distinct sequences elements from a mouse Id1 promoter [15]. The addition of BMP2 (0.5-100 ng/ml) to the transfectants resulted in a dose-dependent increase in luciferase activity in the cell lysates. This new assay was 100-fold more sensitive than the classical alkaline phosphatase (ALP) activity assay (0.5-1 vs. 50-100ng/ml, respectively) as well as much more rapid (24 h vs. 3-6 days, respectively, of BM P treatment). This new assay is specific to BMPs (BMP-2, BMP-4, and BMP7) as evidenced by its relative insensitivity to TGF beta 1, bFGF, and VEGF. Because of its BMP specificity, this rapid, sensitive, nonradioactive, and easily performed assay could be used in monitoring the biological activity of BMP and, eventually, as a cell-based screening assay to identify and evaluate molecules that modulate BMP signaling in cells. (c) 2005 Elsevier Inc. All rights reserved.