Zhang Guo-Jun, Kaelin William G
Howard Hughes Medical Institute, Dana-Farber Cancer Institute and Brighan and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.
Methods Enzymol. 2005;399:530-49. doi: 10.1016/S0076-6879(05)99036-8.
Optical imaging of reporter molecules such as firefly luciferase has become a popular method of tracking and visualizing cells in living animals. Many biological processes involve ubiquitin ligases, which target specific proteins for destruction under specific sets of conditions. Importantly, the motifs recognized by different ubiquitin ligases are often modular and can be used to target foreign proteins for destruction in cis. We recently fused the Cdk inhibitor p27, which is polyubiquitylated by a Skp2-containing ubiquitin ligase if phosphorylated by cdk2 to firefly luciferase. The resulting fusion protein, p27-Luc, was induced by cdk2 inhibitors in living cells grown in culture or in nude mice. This article describes protocols for validation of p27-Luc in cell culture using siRNA against cdk2 (or its partner cyclin A) and for imaging cells producing p27-Luc grown in transparent hollow fibers after treatment with cdk2 inhibitory drugs in vivo. These approaches should be generalizable to other ubiquitin-ligase substrate pairs.
诸如萤火虫荧光素酶等报告分子的光学成像已成为在活体动物中追踪和可视化细胞的一种常用方法。许多生物过程涉及泛素连接酶，这些酶在特定条件下会将特定蛋白质作为靶标进行降解。重要的是，不同泛素连接酶识别的基序通常是模块化的，可用于在顺式作用中靶向异源蛋白质进行降解。我们最近将细胞周期蛋白依赖性激酶（Cdk）抑制剂p27与萤火虫荧光素酶融合，p27如果被Cdk2磷酸化，会被含Skp2的泛素连接酶多聚泛素化。所得的融合蛋白p27-Luc在培养的活细胞或裸鼠中可被Cdk2抑制剂诱导产生。本文介绍了在细胞培养中使用针对Cdk2（或其伴侣细胞周期蛋白A）的小干扰RNA（siRNA）验证p27-Luc的方案，以及在体内用Cdk2抑制药物处理后，对在透明中空纤维中生长的产生p27-Luc的细胞进行成像的方案。这些方法应可推广至其他泛素连接酶-底物对。
