Development and use of systems to study estrogen action

描述(由申请人提供) 鉴定含有功能性雌激素的组织和细胞 受体(ERs)，转基因小鼠，Erin(雌激素受体作用指示剂)， 是通过结合一个内质网活动的报告程序而开发的 与报告基因连锁的雌激素反应启动子的转基因 β-半乳糖苷酶。初步分析ERIN小鼠显示内质网活跃 各种组织，包括子宫、脑下垂体和肝脏。Erin鼠标将会 被用来帮助识别在单个细胞中调节的基因 其中ER-配体，雌激素和选择性雌激素受体调节剂 (2)新型内质网共调节蛋白。通过 随着报告基因在ER配体存在下的表达，细胞 含有活化内质网的细胞将用荧光激活细胞分离 分类。首先，将从捕获的细胞中分离出RNA，并将cRNA 标记并用于探针小鼠寡核苷酸基因阵列的鉴定 特定类别内质网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 以特定的内质网配体复合体为诱饵，然后进行蛋白质组层析 用混合多肽测序鉴定相互作用的蛋白质 直接和急诊室联系。预计蛋白质的鉴定 对不同ER配体的细胞选择性作用的重要性将部分 解释ER-配体细胞的特异性并促进鉴定 新一代SERM通过筛选促进一类 ER-辅因子与另一个因子的相互作用。 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，和我一起去教员的位置。因此，我 选择这个项目作为我可以围绕它开发一个独立的 职业生涯。我觉得我离实现这个目标已经走了一半。然而，一个 额外的训练期将使我能够延长这项令人兴奋的研究 计划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，以促进这些事业和研究 目标以下列具体方式：1)申请人的导师Dr。 在雌激素领域，麦克唐奈是一位卓有成效的研究人员。 受体作用，2)在基因组学、蛋白质组学方面有专长的五名合作者， 哺乳动物的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 杜克大学校园里有设备，还有一份新的双周刊 基因组学和蛋白质组学系列研讨会将提供最新信息 以及一个论坛，讨论拟议的研究项目。

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.