Plasmid Delivery & Expression in Embryonic Eye Tissues

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): The study of signal transduction depends on understanding protein-protein interactions within the cell or tissue. The goal of this proposal is to develop pilot data, reagents and procedures that will provide supporting data for a R01 application. The immediate goal of this application is to develop the use of fluorescence resonance energy transfer (FRET) to study, in an intact whole embryonic eye tissue, the protein-protein interactions occurring during signal transduction. To accomplish this goal we will modify (1) expressing vectors that fluorescently tag target proteins of interest, and (2) develop methods for transfection of these vectors into intact eye tissues. Of potentially interacting target proteins, one will be expressed with a cyan fluorescent protein (CFP) tag, and the other expressed with a yellow fluorescent protein (YFP) tag. (These are derivatives of green fluorescent protein). A transfected tissue will be stimulated, and signal transduction protein interactions will be followed by fluorescence changes as visualized by FRET microscopy. Two advances to existing technology are proposed. First, designing and testing constructs that contain two fusion proteins in a single vector (two-in-one) in cultured cells. Several alternative designs for the two-in-one vector system are proposed including using (1) an internal enzyme cleavage site, (2) a vector with an internal ribosomal entry site (IRES), or (3) having two mini genes in tandem. The proteins produced by the vectors will be tested for protein function and energy transfer in single cells before using them in whole tissues. Secondly, the two fusion proteins will be transfected and analyzed in whole embryonic tissues. This will require transfecting primary tissues. Several alternative approaches have been proposed including commercial permeabilization agents, electroporation or viral infections. Transfection effectiveness will be assessed with flow cytometry, GST pull-down assays, and Western blots. The protein-protein interactions between Rho and ROCK with all appropriate controls will be the first test pair for FRET in whole corneal epithelia. Once the procedures are developed the number of probes will be expanded to study other upstream and down stream proteins in the Rho activation-signaling pathway in future grant applications. This is a transfer of an established technique for tissue culture or single cells into whole embryonic tissues. The development of these techniques will have a significant impact on future research in ocular development because direct protein-protein interactions may be visualized. This project has a high risk because neither "two-in-one" vectors, nor transfection into whole tissues has been accomplished.