Core--Functional Genomics

描述：这个新设施核心的目标是提供中心 研究人员使用广泛的可靠和有效的检测方法来分析变化 并为基因表达的储存和 分析大量数据。本核心的一个具体目标是提供 对中心成员进行高通量测定（检查全基因组变化），例如 作为cDNA微阵列和基因表达系列分析（SAGE），以及 中等通量测定（检查数十至数百个基因），例如 基因表达快速分析（PCR）和实时PCR。其他目标是 交叉验证这些技术，以获得和维持适当的cDNA， 引物库，并获得和/或开发生物信息学支持。 赠款申请指出，该核心计划提供以下内容 对中心成员的支持：1）使用外部和内部数据库; 2)培训和支持SAGE，微阵列，实时PCR; 3)经验证的实时PCR引物和探针，PCR引物和接头， 微阵列芯片（最初是小鼠和大鼠，如果是，则添加鱼和人， 需要/保证）和荧光探针，用于测序的SAGE克隆; 4）QC 检查cDNA制备; 5）储存全局表达数据; 6）交钥匙 SAGE，cDNA微阵列，PCR和实时PCR实验，与成员 提供cDNA制备物和核心最终数据。此外，本发明还提供了一种方法， 本核心计划建立各种数据的可靠性和有效性， 分析;开发方法，用于分析从 显微切割;开发和维护一个引物和接头库 和实时PCR分析库;并开发计算机方法， 自动化实验设计，数据收集，分析和演示 全局表达式数据。 该核心将建立在麦克劳德博士和Aldaz博士的现有专业知识基础上 还有迪乔瓦尼麦克劳德博士是该技术的主要发明者。的愤怒 技术已被用于检测小鼠中基因表达的变化， 转基因小鼠中E2F1基因过表达的角质形成细胞，与 在野生型小鼠中的表达。它也被用来检查DNA损伤 人乳腺上皮细胞HME 87对BPDE处理的反应以及 用BPDE或DMBA处理的小鼠皮肤。这项技术已经 转让给其他CRED成员和外部生物技术公司， 正在开发的商业软件。阿尔达斯博士成功地利用 SAGE技术，以评估MCF-7细胞中基因表达的变化， 雌激素治疗Aldaz博士已经从正常的 乳腺组织、乳腺肿瘤和乳腺癌细胞系。乳腺癌 和正常乳腺的文库已经捐赠给了癌症基因组解剖学 项目的SAGE数据库。DiGiovanni博士使用Clontech低密度cDNA 阵列和Genome Systems Gem-1高密度cDNA阵列成功地 描述转基因动物的特征。

Description: The objective of this new Facility Core is to provide Center investigators a wide range of reliable and validated assays to analyze changes in gene expression and to provide informatics support for the storage and analysis of large arrays of data. A specific aim of this Core is to provide to Center members high throughput assays (examining genome-wide changes), such as cDNA microarrays and serial analysis of gene expression (SAGE), as well as the medium throughput assays (examining tens to hundreds of genes), such as rapid analysis of gene expression (RAGE) and real-time PCR. Other aims are to cross validate these technologies, to obtain and maintain appropriate cDNA and primer libraries, and to obtain and/or develop bioinformatics support. The grant application stated that this Core plans to provide the following support to Center members: 1) use of external and internal database; 2) training and support for SAGE, RAGE, microarrays, and real-time PCR; 3) validated real-time PCR primers and probes, RAGE primers and linkers, microarray chips (mouse and rat initially, add fish and human if needed/warranted) and fluorescent probes, SAGE clones for sequencing; 4) QC check for cDNA preparations; 5) storage of global expression data; 6) turnkey SAGE, cDNA microarray, RAGE, and real-time PCR experiments, with the members providing the cDNA preparations and the Core the final data. In addition, this Core plans to establish the reliability and validity of the various assays; develop methods for assaying small samples obtained from microdissection; develop and maintain a library of RAGE primers and adapters and a library of real-time PCR assays; and develop computer methods to automate experimental design, data gathering, analysis and presentation of global expression data. This Core will build upon the pre-existing expertise of Drs. MacLeod, Aldaz and DiGiovanni. Dr. MacLeod is the primary inventor of RAGE. The RAGE technology has been used to detect changes in gene expression in murine kereatinocytes overexpressing the E2F1 gene in transgenic mice, compared to the expression in wildtype mice. It has also been used to examine DNA damage response of human mammary epithelial cell HME87 to treatment with BPDE and of mouse skin treated with either BPDE or DMBA. This technology has been transferred to other CRED members and to an external biotech company, with a commercial RAGE software under development. Dr. Aldaz has successfully used the SAGE technology to evaluate changes in gene expression in MCF-7 cells upon treatment with estrogen. Dr. Aldaz has generated SAGE libraries from normal breast tissues, breast tumors, and breast cancer cell lines. Breast cancer and normal breast libraries have been donated to the Cancer Genome Anatomy Project's SAGE database. Dr. DiGiovanni has used Clontech low density cDNA array and Genome Systems Gem-1 higher density cDNA array successfully to characterize transgenic animals.