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

该项目将开发新技术，用于测量真核生物体内的整体基因表达，这将允许分析复杂组织中包含的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。第一种是使用维多利亚水母的绿色荧光蛋白以细胞类型特异性的方式标记细胞核，所述绿色荧光蛋白与细胞核内积累的蛋白质特异性地融合。这将通过产生转基因植物来完成，其中GFP的核靶向形式的产生在细胞类型特异性启动子的控制下。将通过荧光显微镜验证特异性标记的细胞核的存在，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，并与DNA微阵列杂交。概念验证将首先涉及使用组成型活性启动子验证策略的不同技术步骤，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聚核糖体。这将通过单个核糖体蛋白的表位标记来完成，所述单个核糖体蛋白的表达由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，并将相关的mRNA用于微阵列杂交。如前所述，概念验证实验将涉及用组成型启动子验证该策略的技术方面，然后使用具有确定的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。该项目的预期成果有三个方面：一个分子工具包（试剂、重组DNA分子、植物系），如何使用该工具包检查活生物体内基因表达的相关说明，以及为验证该方法而进行的实验结果。所有成果将及时免费分发给科学界。将应要求向感兴趣的个人提供分子工具包。实验方法和结果的说明将张贴在项目网站上，并酌情在科学文献中发表。这些方法被设计为在范围上完全通用，并且应该可转移到其他真核生物，包括植物界以外的真核生物。可行性：我们建议将所有重组克隆免费分发给学术界。克隆人接受者将被要求支付快递公司的运输费。如果某些复制品被证明非常受欢迎，我们保留对它们的分发收取象征性费用的权利。知识产权问题将根据两个机构的要求处理。有关技术转让官员目前正在进行谈判，以建立一个适当的机制，发展可能从研究中产生的知识产权。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）。由于联合大学是国家科学基金赠款的牵头机构，因此也期望联合大学成为商业化的管理机构。正在根据现有的统一通信文件制定独立审计员模板。 相关的特征是发明的50：50分割（以简化程序），以及最短的出版延迟时间以允许寻求专利保护（目前设想为60天）。我们将采用通用标准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/