CORE--TECHNICAL SUPPORT

技术核心将提供三大功能，协助项目编制小组的四个项目。这些是蛋白质纯化，蛋白质表达分析和基因表达分析。下文概述了这些活动的组成部分，其中包括支助人员和设备。技术核心将促进每个项目的进展，如下所述，以及组成计划项目的五个实验室之间的互动。1.蛋白质纯化：FPLC。Perkins目前有一个用于蛋白质纯化的FPLC，这将成为技术支持核心的一部分。这将被Krause的项目（项目2）用于纯化与CD 34启动子上游调控区结合的因子。它也将被Weissman的项目（Specific Aim 4）用于鉴定与嗜中性粒细胞活化下调的基因上游调控序列结合的蛋白质。由于FPLC已经在现场，我们只要求额外的列是必要的。2.蛋白表达分析A. 2d-GELS：设备、供应品、技术员（郑博士）和Melanie II软件B。MALDI-MS：技术员）Edward Papacoda）。本PPG的一个关键的压倒一切的主题是分析骨髓生成过程中造血细胞的分子解剖学，包括初始阶段（项目2），以及正常和EVI-1表达细胞的诱导成熟（项目3），以及对E. coli（Project 4）。这些项目将检查mRNA水平（见下一节）和蛋白质水平的变化。分子生物学中最令人兴奋的新发展之一是通过胰蛋白酶消化和质谱法识别蛋白质的能力，使用MALDI-MS（基质辅助激光解吸/电离质谱法）结合Prosite软件搜索蛋白质和DNA序列数据库。这允许使用低至10 fmol的蛋白质来鉴定，这小于1 ng的50，000道尔顿蛋白质（相当于银染凝胶上可检测的下限）。同时，用于蛋白质的2D凝胶分析的硬件和软件已经改进，以允许蛋白质的可再现分级分离，以及不同凝胶的详细比较以确定定量差异。我们打算充分利用这些进步，并要求提供资金，以购买设备、用品和技术支持，使我们能够做到这一点。这一核心设施将积累的信息和专门知识将大大有利于本项目规划补助金中的所有项目，并为其提供令人信服的支持。3.基因表达分析A.亲和基因芯片技术B.差分显示3.序列分析mRNA差异分析项目的共同主题将由技术核心的三个关键组成部分支持。Affyscore基因芯片分析系统允许评估数千种不同小鼠基因的表达水平。目前，在四个芯片上有6,500个小鼠基因。这些基因作为微阵列存在于硅芯片上，并使用标准DNA合成化学以及计算机芯片制造中使用的掩蔽技术原位合成。使用感兴趣的mRNA作为模板，将阵列与荧光标记的cDNA杂交，并通过扫描激光“读取”杂交。Affytron系统由五个组件组成：GeneChip探针阵列、GeneChip Fluidics Station 400、Hewlett-Packard GeneArray扫描仪和GeneChip探针阵列、基因芯片软件。总之，该系统使杂交、洗涤以及数据捕获和分析中的许多步骤自动化。

The Technical Core will provide three broad functions that will assist the four Projects of the PPG. These are Protein Purification, Protein Expression Analysis, and Gene Expression Analysis. The components of these are outlined below, and include both support personnel and equipment. The Technical Core will facilitate the progress of each project as described below, as well ss interaction between the five labs comprising the Program Project. 1. Protein purification: FPLC. The Perkins currently has an FPLC for protein purification, and this will become part of the Technical Support Core. This will be utilized by Krause's project (Project 2) to purify the factor that binds to the upstream regulatory region of the CD34 promoter. It will also be used by Weissman's project (Specific Aim 4) to identify the proteins that bind to upstream regulatory sequences of genes that are down- regulated on neutrophil activation. Since the FPLC is already on site, we only requesting additional columns that are needed. 2. Protein expression analysis A. 2d-GELS: Equipment,, supplies, technician (Dr. Zheng) and Melanie II software B. MALDI-MS: Technician )Edward Papacoda). A key overriding theme to this PPG is the analysis of the Molecular Anatomy of hematopoietic cells during the process of myelopoiesis, including the initial stages (Project 2), as well as in induced maturation in normal and EVI-1-expressing cells (Project 3), and in response to E. coli (Project 4). These projects will examine changes in both the mRNA level (see next section) and the protein level. One of the most exciting new developments in molecular biology is the ability to identify proteins by tryptic digest and mass spectrometry, using the MALDI-ms (matrix-assisted laser desorption/ionization mass spectrometry) in conjunction with the Prosite software for searching protein and DNA sequence databases. This has allowed the identification of proteins using as little as 10 fmol, which is less than 1 ng of a 50,000 dalton protein (equivalent to the lower limit of what is detectable on silver-stained gels). In parallel, hardware and software for 2D gel analysis of proteins has improved, to allow reproducible fractionation of proteins, and detailed comparison of different gels to identify quantitative differences. We intend to take full advantage of these advances, and are requesting money for equipment, supplies, and technical support to enable us to do this. The cumulative accrual of information and expertise that will occur with this core facility will substantially benefit all of the projects in this PPG, and provides a cogent argument in its support. 3. Gene expression analysis A. Affymetrix gene chip technology B. Differential Display 3. Sequence analysis The common theme among the projects of mRNA difference analysis will be supported by three key components of the Technical Core. The Affymetrix GeneChip analysis system allows the assessment of expression levels for thousands of different mouse genes. At present, 6,500 mouse genes are available on four chips. These genes are present as microarrays on a silica chip and are synthesized in situ using standard DNA synthesis chemistry as well as masking technology utilized in computer chip manufacturing. The arrays are hybridized with fluorescently labeled cDNA, using the mRNA of interest as a template, and the hybridization is "read" by a scanning laser. The Affymetrix system consists of five components: the GeneChip Probe Array, GeneChip Fluidics Station 400, Hewlett-Packard GeneArray Scanner, and GeneChip Probe Array, Gene Chip Software. Together, this system has automated many steps in the hybridization, washing, and data capture and analysis.