Scientific

科学核心的长期目标是提供杰出的，最先进的，具有成本效益的 支持该计划所有五个项目的核心设施和服务。核心B由一种蛋白质组成 表达和纯化设施，以及其他共享设施，包括暗室，PhosphorImager， 超浓缩机和液氮冷冻机。核心还将支持逆转录病毒的生成 表达不同的shRNA和miRNA表达的微阵列分析。 PhosphorImager将用于成像和各种结果的定量，包括预充伸展 分析、凝胶迁移率变化和Westem印迹。蛋白质表达和纯化设施 由振荡培养箱、离心机、FPLC和蛋白表达载体组成。它是用来表达 并纯化在每个项目中研究的蛋白质。在一项新的服务中，核心还将产生 用于各种项目的shRNA构建体。这将包括设计靶向特定基因的shRNA， 将插入片段克隆到逆转录病毒载体中，并产生高滴度逆转录病毒原液。核心也将 通过购买用于分析的试剂盒来支持Taqman microRNA微阵列设置的使用。一个 还将购买Amaxa Nucleofector转染设备供项目使用。核还 拥有用于储存细胞系的液氮冷冻机和凝胶记录系统。此外，本发明还提供了一种方法， 由监督核心的人员向所有成员实验室提供专业知识。这个科学核心是 这对该计划的成功至关重要，并促进成员实验室之间的互动。

The long-term objective of the Scientific Core is to provide outstanding, state-of-the-art, cost-effective Core facilities and services that support all five projects of the Program. Core B is composed of a protein expression and purification facility, as well as other shared facilities including a darkroom, Phosphorlmager, ultracentrifuge, and liquid nitrogen freezers. The Core will also support the generation of retroviruses expressing variou~ shRNAs and microarray analysis of miRNA expression. The Phosphorlmager will be used for imaging and quantification of various results including primerextension assays, gel mobility shifts, and Westem blots. The protein expression and purification facility consists of a shaking incubator, a centrifuge, an FPLC, and protein expression vectors. It is used to express and purify proteins that are studied in each of the projects. In a new service, the Core will also generate shRNA constructs for the various projects. This will include design of shRNAs that target specific genes, cloning the inserts into retrovirus vectors, and generation of high-titer retrovirus stocks. The Core will also support the use of a Taqman microRNA microarray set-up by purchasing cassettes for the analysis. An Amaxa Nucleofector transfection apparatus will also be purchased for use by the projects. The Core also possesses liquid nitrogen freezers for the storage of cell lines and a gel documentation system. In addition, expertise is provided to all member laboratories by personnel who oversee the Core. This Scientific Core is essential to the success of the Program, and facilitates interaction between member laboratories.