Technology Development: Novel Techniques for Discovery of Patterns of Gene Regulation Within Complex Eukaryotic Tissues.

该项目将开发测量真核生物内全球基因表达的新技术，这将使分析复杂组织中包含的不同细胞类型的个体贡献成为可能。该项目包括一系列概念验证实验，主要使用拟南芥。我们将采用两种策略。第一种是使用Aequorea Victoria的绿色荧光蛋白以特定细胞类型的方式标记细胞核，该蛋白翻译后融合到细胞核内积累的蛋白质上。这将通过生产转基因植物来实现，在转基因植物中，GFP的核靶向形式的产生受到细胞类型特定启动子的控制。特殊标记的核的存在将通过荧光显微镜进行验证，并使用荧光激活分选从细胞匀浆中纯化单个荧光核。然后从细胞核中提取RNA，并将其与DNA微阵列杂交。概念验证将首先涉及使用具有结构性活性的启动子对该战略的不同技术步骤进行验证，然后将涉及使用已知细胞特性的启动子定义的细胞类型中的全球基因表达分析。第二种策略是以类似的细胞类型特异性方式标记多聚核糖体。这将通过对单个核糖体蛋白进行表位标记来实现，这些蛋白的表达受到具有细胞类型特定表达模式的启动子的调节。然后将制备多核糖体，并将相关的mRNA用于微阵列杂交。和以前一样，概念验证实验将涉及用构成启动子验证这一策略的技术方面，然后使用定义了细胞特异性的启动子来绘制这些细胞内的全球基因表达模式。这个项目的预期结果有三个方面：一个分子工具包(试剂、重组DNA分子、植物品系)，如何使用这个工具包检查活生物体内的基因表达的相关说明，以及为验证该方法而进行的实验的结果。所有成果将及时自由传播给科学界。分子工具包将应要求提供给感兴趣的个人。这些方法和实验结果的说明将公布在项目网站上，并酌情发表在科学文献上。这些方法被设计成完全通用的范围，并且应该可以转移到其他真核生物，包括植物界以外的生物。交付成果：我们建议将所有重组克隆免费分发给学术界。克隆接受者将被要求支付快递公司的交通费。如果某些克隆被证明异常受欢迎，我们保留对其分发收取象征性费用的权利。知识产权问题将根据这两个机构的要求处理。目前，有关技术转让官员之间正在进行谈判，以建立一个适当的知识产权开发机制，该机制可能会从研究中发展出来。预计UA和UCR将在适当时候就知识产权商业化达成一项机构间协议(IIA)。由于UA是NSF赠款的牵头机构，因此也预计UA应该成为商业化目的的管理机构。正在根据现有的统一通信文件开发IAA模板。相关的特征是50：50的发明分割(以简化程序)，以及允许申请专利保护的最短发布延迟时间(目前设想为60天)。我们将采用共同标准来获取和存档所有微阵列数据，如http://plantgenome.sdsc.edu/AwardeesMeeting/Bioinformatics_and_Databases/所述

This project will develop novel techniques for measurement of global gene expression within eukaryotic organisms which will permit analysis of the individual contributions of the different cell types contained within complex tissues. The project comprises a series of proof-of-concept experiments primarily using Arabidopsis thaliana. Two strategies will be employed. The first is to label nuclei in a cell type-specific manner using the Green Fluorescent Protein of Aequorea victoria translationally fused to proteins that accumulate within the nucleus. This will be done by producing transgenic plants in which the production of a nuclear targeted form of GFP is under the control of cell type specific promoters. The presence of specifically labeled nuclei will be verified by fluorescence microscopy, and the individual fluorescent nuclei purified from cellular homogenates using fluorescence activated sorting. RNA will then be extracted from the nuclei and will be hybridized to DNA microarrays. Proof-of-concept will first involve validation of the different technical steps of the strategy, using promoters that are constitutively active, and then will involve analysis of global gene expression within the cell types defined using promoters of known cellular specificities. The second strategy is to label polyribosomes in an analogous cell-type specific manner. This will be done through epitope tagging of individual ribosomal proteins whose expression is regulated by promoters having cell type-specific patterns of expression. Polyribosomes will then be prepared and the associated mRNA employed for microarray hybridization. Proof-of-concept experiments, as before, will involve validation of the technical aspects of this strategy with constitutive promoters, followed by use of promoters having defined cellular specificities to chart global gene expression patterns within these cells. The expected outcomes of this project are three-fold: a molecular toolkit (reagents, recombinant DNA molecules, plant lines), the associated descriptions of how to use this toolkit for examination of gene expression within living organisms, and the results of the experiments that are done to validate the methodology. All outcomes will be freely disseminated to the scientific community in a timely manner. The molecular toolkits will be provided to interested individuals on request. The descriptions of the methods and the results from the experiments will be posted to the project website and, as appropriate, will be published in the scientific literature. The methods are designed to be entirely general in scope, and should be transferable to other eukaryotic organisms, including those of other than the plant kingdom. Deliverables:We propose to distribute all recombinant clones to the academic community free of charge. Clone recipients would be expected to pay transportation charges for express carriers. Should specific clones prove inordinately popular, we reserve the right to institute nominal fees for their distribution.Intellectual Property issues will be handled according to the requirements of the two institutions. Negotiations are currently underway between the relevant technology transfer officers to establish an appropriate mechanism for development of intellectual property which might develop from the research. It is expected that UA and UCR will enter into an interinstitutional agreement (IIA) at the appropriate time for the commercialization of IP. As UA is the lead institution on the NSF grant, it is also expected that UA should be the managing institution for purposes of commercialization. An IAA template is being developed based on an existing UC document. Relevant features are a 50:50 split of inventions (to simplify procedures), and a minimal time for delay of publications to permit patent protection to be sought (currently envisaged to be 60 days).We shall employ common standards for acquiring and archiving all microarray data as described at: http://plantgenome.sdsc.edu/AwardeesMeeting/Bioinformatics_and_Databases/