Plasmid Delivery & Expression in Embryonic Eye Tissues

描述（由申请人提供）：信号转导的研究取决于对细胞或组织内蛋白质-蛋白质相互作用的理解。 本提案的目标是开发试验数据、试剂和程序，为R 01应用提供支持数据。本申请的直接目标是开发使用荧光共振能量转移（FRET）研究，在一个完整的胚胎眼组织中，在信号转导过程中发生的蛋白质-蛋白质相互作用。为了实现这一目标，我们将修改（1）表达载体，荧光标记的目标蛋白，和（2）开发这些载体转染到完整的眼组织的方法。 在潜在相互作用的靶蛋白中，一种将用青色荧光蛋白（CFP）标签表达，另一种用黄色荧光蛋白（YFP）标签表达。 （这些是绿色荧光蛋白的衍生物）。 将刺激转染的组织，并且信号转导蛋白相互作用之后将是荧光变化，如通过FRET显微镜观察到的。 提出了现有技术的两个进步。 首先，在培养的细胞中设计和测试在单个载体（二合一）中含有两种融合蛋白的构建体。 提出了用于二合一载体系统的几种替代设计，包括使用（1）内部酶切割位点，（2）具有内部核糖体进入位点（IRES）的载体，或（3）具有串联的两个小基因。 由载体产生的蛋白质在用于整个组织之前将在单细胞中测试蛋白质功能和能量转移。 其次，将这两种融合蛋白在整个胚胎组织中转染和分析。 这将需要切除原代组织。 已经提出了几种替代方法，包括商业透化剂、电穿孔或病毒感染。 将使用流式细胞术、GST下拉测定和蛋白质印迹法评估转染有效性。 Rho和ROCK与所有适当对照之间的蛋白质-蛋白质相互作用将是整个角膜上皮中FRET的第一个测试对。 一旦程序开发的探针的数量将扩大到研究其他上游和下游蛋白质在Rho激活信号通路在未来的拨款申请。 这是将已建立的组织培养技术或单细胞转移到整个胚胎组织中。 这些技术的发展将对眼发育的未来研究产生重大影响，因为直接的蛋白质-蛋白质相互作用可以可视化。该项目具有很高的风险，因为“二合一”载体，也没有转染到整个组织已经完成。

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.