Fluorescent visualization of cancer cellsby monitoring cellular polyamines

我们正在开发一种新的方法来可视化癌细胞结合AZ，内源性细胞多胺传感器，和荧光蛋白技术的多胺依赖性移码机制。我们创建了一个多胺传感器构建体，其中AZ 1 mRNA的移码区两侧是两个荧光蛋白（ECFP和Keima-Red）。然而，当将构建体转染到细胞中时，未观察到Keima Red荧光的多胺依赖性增加。因此，我们使用AZ 1 mRNA的整个蛋白质编码区与EGFP基因改进了传感器构建体，所述EGFP基因被插入到假结结构的紧下游。转染该构建体的细胞显示EGFP荧光和移码产物的多胺依赖性增加。细胞中多胺反应的进一步分析继续进行。

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.