Rapid Development of Genetically Encoded FRET Reporters
Rapid Development of Genetically Encoded FRET Reporters
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DOI：
10.1021/cb100402n
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发表时间：
2011-07-01
影响因子：
4
通讯作者：
Schultz, Carsten
中科院分区：
文献类型：
--
作者：
Piljic, Alen;de Diego, Inaki;Schultz, Carsten
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报告分子分析策略。多功能和完全正交克隆方法涉及一组多达36个载体，具有各种荧光蛋白FRET对和不同长度的接头。该构建体组成功地应用于两个钙调素结合蛋白，死亡相关蛋白激酶。1（DAPK 1）和钙/钙调蛋白依赖性蛋白激酶II α（Camk 2a）。通过打印质粒DNA阵列和随后的半自动显微镜检查转染的细胞进行克隆分析和报告基因验证。表现最好的DAPK 1和Camk 2a报告分子的表征揭示了在将钙信号翻译成激酶反应方面的显著差异，尽管功能和结构相似。
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 alpha (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.