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

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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/