Development-HTS Assay-Identify Specific Alternative(RMI)

描述（由申请人提供）：该项目的目标是开发强大的分析方法，允许实时监测和定量活细胞中特定的选择性剪接事件，并使其适应针对小化合物文库的高通量筛选（HTS）。替代剪接模式的解除管制通常与几种神经退行性疾病和其他遗传疾病有关，并且在大多数癌症中常见。重新引导剪接事件以纠正此类剪接缺陷或强制表达具有潜在有益作用的替代剪接异构体的能力将为研究特定替代剪接异构体的作用和功能提供有价值的工具，并将为新的治疗方法开辟广阔的可能性。我们计划开发一套minigene结构，其中瞬时转染的荧光报告蛋白的表达将定量地依赖于特定的剪接事件。最初的测试将研究STAT3和BRCA1中可选择的剪接事件，但这种设置将很容易转移到不同的系统。该试验最初将在96孔板上进行，并用于中低通量实验，通过系统地过表达和/或敲低剪接因子，结合底物诱变，来识别和表征调节这些剪接事件的顺式元件和反式因子。多种荧光变体将使用Amersham INCell分析仪以各种组合进行测试，最终开发出一种多色方案，以同时评估单个细胞中的不同剪接事件，以及适当的反筛选和验证程序。该方案将优化到384孔微滴板，将适用于大的小分子文库的HTS。

DESCRIPTION (provided by applicant): The objective of this project is to develop robust assays that will allow real-time monitoring and quantification of specific alternative splicing events in living cells, and to adapt them for high throughput screening (HTS) against libraries of small compounds. Deregulation of alternative splicing patterns is frequently associated with several neurodegenerative and other genetic diseases and is a common occurrence in most cancers. The ability to re-direct splicing events to correct such splicing defects or to force the expression of alternative splicing isoform with potential beneficial effects would provide a valuable tool to investigate the roles and functions of specific alternative splicing isoforms, and it will open wide possibilities for novel therapeutic approaches. We plan to develop a set of minigene constructs in which the expression of a transiently transfected fluorescent reporter protein will quantitatively depend on a specific splicing event. The initial testing will investigate alternative splicing events in STAT3 and BRCA1, but the setup will be readily transferable to different systems. The assay will be initially performed in 96 well plates and used in low-to-medium throughput experiments to identify and characterize cis-elements and trans acting factors that regulate these splicing events, by means of systematic over-expression and/or knockdown of splicing factors combined with substrate mutagenesis. Multiple fluorescent variants will be tested in various combinations using the Amersham INCell analyzer, to eventually develop a multicolor protocol to simultaneously assess different splicing events in individual cells, along with appropriate counter screenings and validation procedures. The protocol will be optimized to 384 well microtiter plates and will be suitable to HTS of large small molecule libraries.