Heydorn Arne, Lundholt Betina K, Praestegaard Morten, Pagliaro Len
Fisher BioImage ApS, Søborg, Denmark.
Methods Enzymol. 2006;414:513-30. doi: 10.1016/S0076-6879(06)14027-6.
Redistribution technology is a cell-based assay technology that uses protein translocation as the primary readout for the activity of cellular signaling pathways and other intracellular events. Protein targets are labeled with the green fluorescent protein, and stably transfected cell lines are generated. The assays are read using a high-throughput, optical microscope-based instrument, several of which have become available commercially. Protein translocation assays can be formatted as agonist assays, in which compounds are tested for their ability to promote protein translocation, or as antagonist assays, in which compounds are tested for their ability to inhibit protein translocation caused by a known agonist. Protein translocation assays are high-content, high-throughput assays primarily used for profiling of lead series, primary screening of compound libraries, and as readouts for gene-silencing studies using siRNAs. This chapter describes two novel high-content Redistribution assay technologies: (1) The p53:hdm2 GRIP interaction assay, in which one high-content image feature is used for detection of primary hits, whereas a different feature is used to deselect compounds with unwanted mode of action, and (2) application of siRNAs to Redistribution assays, exemplified by knockdown of Akt isoforms in a FKHR translocation assay reporting on the PI3 kinase signaling pathway.
再分布技术是一种基于细胞的检测技术，它利用蛋白质转位作为细胞信号通路及其他细胞内事件活性的主要读出指标。蛋白质靶点用绿色荧光蛋白标记，并构建稳定转染的细胞系。检测通过基于光学显微镜的高通量仪器进行，其中有几种已商业化。蛋白质转位检测可设计为激动剂检测，即测试化合物促进蛋白质转位的能力，也可设计为拮抗剂检测，即测试化合物抑制已知激动剂引起的蛋白质转位的能力。蛋白质转位检测是高内涵、高通量检测，主要用于先导化合物系列分析、化合物库的初步筛选以及作为使用小干扰RNA进行基因沉默研究的读出指标。本章描述了两种新型高内涵再分布检测技术：（1）p53:hdm2 GRIP相互作用检测，其中一种高内涵图像特征用于检测主要命中物，而另一种不同特征用于排除具有不良作用模式的化合物；（2）小干扰RNA在再分布检测中的应用，以在报告PI3激酶信号通路的FKHR转位检测中敲低Akt亚型为例。
