Development and use of systems to study estrogen action

开发和使用研究雌激素作用的系统

• 批准号: 6419164
• 负责人: SUSAN C NAGEL
• 金额: $ 3.04万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6419164
• 关键词: affinity chromatography; estrogen receptors; estrogens; functional /structural genomics; gene expression; genetically modified animals; hormone regulation /control mechanism; laboratory mouse; microarray technology; protein sequence; proteomics; receptor expression

DESCRIPTION (provided by applicant) To identify tissues and cells that contain functionally active estrogen receptors (ERs), a transgenic mouse, ERIN (estrogen receptor action indicator), was developed that functions as a reporter of ER activity by incorporating a transgene with an estrogen responsive promoter linked to the reporter gene beta-galactosidase. Initial analysis of ERIN mice demonstrated active ER in a variety of tissues, including uterus, pituitary, and liver. The ERIN mouse will be used to aid in the identification of 1) genes regulated in individual cells where ER-ligands, both estrogens and selective estrogen receptor modulators (SERMs), exert agonist activity and 2) novel ER comodulatory proteins. By following expression of the reporter gene in the presence of ER-ligands, cells containing activated ER will be isolated using fluorescence-activated cell sorting. First, RNA will be isolated from captured cells, and cRNA will be labeled and used to probe mouse oligonucleotide gene arrays to identify fingerprints of gene expression for specific classes of ER-ligands. Second, proteins will be isolated from sorted cells and subjected to affinity chromatography using specific ER-ligand complexes as bait, followed by proteome analysis using mixed peptide sequencing to identify proteins that interact directly with ER. It is anticipated that the identification of proteins important for the cell selective actions of different ER-ligands will partially explain ER-ligand cell specificity and facilitate the identification of the next generation SERMs by screening for compounds which facilitate one class of ER-cofactor interaction over another. Before this project was initiated, my mentor and I agreed that I could carry my work with the ERIN mice forward with me to a faculty position. Consequently, I selected this project as one around which I could develop an independent career. I feel that I am halfway toward accomplishing this goal. However, an additional period of training would allow me to extend this exciting research project into the new areas of genomics and proteomics. Duke University provides an excellent research environment to further these career and research objectives in the following specific ways: 1) the applicant's mentor, Dr. McDonnell, is an accomplished and productive researcher in the area of estrogen receptor action, 2) five collaborators with expertise in genomics, proteomics, and mammalian physiology and genetics have agreed to aid in the proposed research and training program, 3) a DNA microarray core facility and other key equipment are available on the Duke University campus, and 4) a new biweekly seminar series in genomics and proteomics will provide the latest information and a forum to discuss the proposed research project.

描述（由申请人提供） 识别含有功能活性雌激素的组织和细胞 受体（ER），转基因小鼠，ERIN（雌激素受体作用指示器）， 被开发出来，通过合并一个 ER 活动的报告者 具有与报告基因连接的雌激素响应启动子的转基因 β-半乳糖苷酶。对 ERIN 小鼠的初步分析表明，ERIN 小鼠中的活性 ER 多种组织，包括子宫、垂体和肝脏。 ERIN 鼠标将 用于帮助识别 1) 单个细胞中调控的基因 其中 ER 配体，包括雌激素和选择性雌激素受体调节剂 (SERM)，发挥激动剂活性和 2) 新型 ER 共调节蛋白。经过 在 ER 配体存在的情况下报告基因表达后，细胞 使用荧光激活细胞分离含有激活的 ER 排序。首先，从捕获的细胞中分离RNA，并将cRNA 标记并用于探测小鼠寡核苷酸基因阵列以识别 特定类别 ER 配体的基因表达指纹。第二， 蛋白质将从分选的细胞中分离出来并进行亲和力分析 使用特定 ER-配体复合物作为诱饵的色谱法，然后进行蛋白质组分析 使用混合肽测序来识别相互作用的蛋白质进行分析 直接与急诊室联系。预计蛋白质的鉴定 对于不同 ER 配体的细胞选择性作用很重要 解释 ER 配体细胞特异性并促进鉴定 下一代SERM，通过筛选促进一类化合物的化合物 ER-辅因子之间的相互作用。 在这个项目开始之前，我和我的导师同意我可以携带我的 与 ERIN 小鼠一起工作，并与我一起晋升到教职职位。因此，我 选择这个项目作为我可以围绕它开发一个独立的项目 职业。我觉得我已经实现了这个目标的一半了。然而，一个 额外的培训时间将使我能够扩展这项令人兴奋的研究 项目进入基因组学和蛋白质组学的新领域。杜克大学提供 良好的研究环境可以促进这些职业和研究的发展 具体通过以下方式实现目标：1）申请人的导师Dr. 麦克唐纳 (McDonnell) 是雌激素领域一位卓有成就且富有成效的研究员 受体作用，2）五名具有基因组学、蛋白质组学专业知识的合作者， 哺乳动物生理学和遗传学已同意协助拟议的 研究和培训计划，3) DNA 微阵列核心设施和其他关键设施 杜克大学校园提供设备，4) 新的双周刊 基因组学和蛋白质组学系列研讨会将提供最新信息 以及讨论拟议研究项目的论坛。