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

该项目将开发用于测量真核生物体内整体基因表达的新技术，这将允许分析复杂组织中不同细胞类型的个体贡献。该项目包括一系列主要使用拟南芥的概念验证实验。将采用两种策略。第一种方法是利用维多利亚Aequorea的绿色荧光蛋白翻译融合到细胞核内积累的蛋白质中，以细胞类型特异性的方式标记细胞核。这将通过生产转基因植物来实现，在转基因植物中，一种核目标形式的绿色荧光蛋白的产生是在细胞类型特异性启动子的控制下进行的。特异性标记细胞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细胞核。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对该策略的不同技术步骤的验证，使用构成活性的启动子，然后将涉及使用已知细胞特异性启动子定义的细胞类型内的全局基因表达分析。第二种策略是以类似的细胞类型特异性方式标记多核糖体。这将通过单个核糖体蛋白的表位标记来完成，其表达受具有细胞类型特异性表达模式的启动子调节。然后制备多核糖体，并将相关mRNA用于微阵列杂交。与之前一样，概念验证实验将涉及使用组成启动子验证该策略的技术方面，随后使用具有定义细胞特异性的启动子来绘制这些细胞内的全局基因表达模式。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来检查活生物体内的基因表达的相关描述，以及为验证方法而进行的实验结果。所有结果将及时向科学界自由传播。分子工具包将按要求提供给感兴趣的个人。方法描述和实验结果将发布在项目网站上，并酌情在科学文献中发表。这些方法的设计在范围上是完全通用的，并且应该可以转移到其他真核生物，包括植物界以外的真核生物。可交付成果：我们建议将所有重组克隆免费分发给学术界。克隆收件人需要支付快递公司的运费。如果特定的克隆被证明非常受欢迎，我们保留对其发行收取象征性费用的权利。知识产权问题将按照两家机构的要求处理。有关的技术转让官员目前正在进行谈判，以建立一个适当的机制，发展可能从研究中发展出来的知识产权。预计UA和UCR将在适当的时候就知识产权的商业化达成机构间协议（IIA）。由于UA是NSF资助的牵头机构，因此也期望UA应该是商业化目的的管理机构。正在根据现有的UC文档开发IAA模板。相关的特点是将发明分成50:50（以简化程序），以及为寻求专利保护而延迟公布的最短时间（目前设想为60天）。我们将采用通用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/