ACS Chem Biol. 2011 Jul 15;6(7):685-91. doi: 10.1021/cb100402n. Epub 2011 Apr 28.
To meet the demand on genetically encoded reporter molecules for live cell imaging, we introduce a new facile combined cloning and FRET reporter analysis strategy. The versatile and fully orthogonal cloning approach involves a set of up to 36 vectors featuring a variety of fluorescent protein FRET pairs and different length linkers. The construct set was successfully applied to two calmodulin-binding proteins, the death-associated protein kinase 1 (DAPK1) and calcium/calmodulin-dependent protein kinase II α (Camk2a). Clone analysis and reporter validation was performed by printing plasmid DNA arrays and subsequent semiautomated microscopy of reversely transfected cells. Characterization of the best performing DAPK1 and Camk2a reporters revealed significant differences in translating calcium signals into kinase responses despite the close functional and structural similarity.
为了满足活细胞成像对基因编码报告分子的需求，我们引入了一种新的简便的组合克隆和 FRET 报告分析策略。这种多功能且完全正交的克隆方法涉及一组多达 36 个载体，具有多种荧光蛋白 FRET 对和不同长度的接头。该构建体集成功应用于两种钙调蛋白结合蛋白，即死亡相关蛋白激酶 1（DAPK1）和钙/钙调蛋白依赖性蛋白激酶 IIα（Camk2a）。通过打印质粒 DNA 阵列并随后对反向转染细胞进行半自动显微镜检查，进行了克隆分析和报告验证。尽管 DAPK1 和 Camk2a 报告器的功能和结构非常相似，但对表现最佳的 DAPK1 和 Camk2a 报告器的特征分析表明，它们在将钙信号转化为激酶反应方面存在显著差异。
