Development and use of systems to study estrogen action

描述（由申请人提供） 鉴定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剂）， 开发了作为ER活性的报告分子的功能， 具有与报告基因连接的雌激素应答启动子的转基因 β-半乳糖苷酶。对ERIN小鼠的初步分析表明， 各种组织，包括子宫、垂体和肝脏。ERIN小鼠将 用于帮助鉴定1）在单个细胞中调节的基因 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）新的ER共调节蛋白。通过 在ER配体存在下表达报告基因后，细胞 将使用荧光激活的细胞分离含有激活的ER的细胞， 分类首先，将从捕获的细胞中分离RNA，并将cRNA从细胞中分离出来。 标记并用于探测小鼠寡核苷酸基因阵列， 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， 使用特异性ER-配体复合物作为诱饵的色谱法，随后进行蛋白质组分析。 使用混合肽测序进行分析以鉴定相互作用的蛋白质 直接与ER。预计蛋白质的鉴定 重要的是，不同ER配体的细胞选择性作用将部分 解释ER-配体细胞特异性，并促进识别 下一代SERMs通过筛选化合物，促进一类 ER-辅因子相互作用。 在这个项目开始之前，我的导师和我同意我可以带着我的 和ERIN老鼠一起工作和我一起去做教职。因此我 我选择了这个项目作为一个我可以开发一个独立的 事业我觉得我已经完成了这个目标的一半。但安 一段额外的训练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 一个良好的研究环境，以促进这些职业和研究 目标在以下具体方式：1）申请人的导师，博士; 麦克唐奈，是一个有成就和生产力的研究领域的雌激素 受体的作用，2）五个合作者在基因组学，蛋白质组学， 哺乳动物生理学和遗传学已经同意帮助提出的 研究和培训计划，3）DNA微阵列核心设施和其他关键 设备在杜克大学校园内可用，4）一个新的双周刊 基因组学和蛋白质组学系列研讨会将提供最新信息 以及一个讨论拟议研究项目的论坛。

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.