Fluorescent visualization of cancer cellsby monitoring cellular polyamines

我们正在开发一种新的方法，通过结合AZ的多胺依赖的移码机制、内源性细胞多胺传感器和荧光蛋白技术来可视化癌细胞。我们构建了一种多胺传感器结构，该传感器含有AZ1mRNA的移码区域和两种荧光蛋白(ECFP和Keima-Red)。然而，当构建的载体被导入细胞时，并没有观察到多胺依赖的Keima-Red荧光增强。因此，我们改进了传感器结构，使用了AZ1mRNA的整个蛋白编码区，并将EGFP基因插入到伪结结构的下游。转染组EGFP荧光和移码产物表达呈多胺依赖性增加。细胞内多胺反应的进一步分析仍在继续。

We are developing a novel method to visualize cancer cells by combining the polyamine-dependent frameshift mechanism of AZ, an endogenous cellular polyamine sensor, and the fluorescent protein techniques. We created a polyamine sensor construct with frameshift region of AZ1 mRNA flanked by two fluorescent proteins (ECFP and Keima-Red). When the construct was transfected into the cells, however, polyamine-dependent increase of Keima-Red fluorescence was not observed. Therefore, we improved the sensor construct using the entire protein coding region of AZ1 mRNA with EGFP gene that is inserted immediately downstream of the pseudoknot structure. Cells transfected with this construct showedpolyamine-dependent increase of EGFP fluorescence and frameshift product. Further analysis of polyamine response in the cells is continued.