Plasmid Delivery & Expression in Embryonic Eye Tissues

描述(由申请人提供)：信号转导的研究依赖于了解细胞或组织内的蛋白质-蛋白质相互作用。这项提案的目标是开发试验数据、试剂和程序，为R01应用提供支持数据。这项应用的直接目标是开发利用荧光共振能量转移(FRET)在完整的胚胎眼组织中研究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)荧光标记目标蛋白的表达载体，以及(2)开发将这些载体导入完整眼睛组织的方法。在可能相互作用的靶蛋白中，一个将用青色荧光蛋白(CFP)标签表达，另一个将用黄色荧光蛋白(YFP)标签表达。(这些是绿色荧光蛋白的衍生品)。转基因组织将受到刺激，信号转导蛋白相互作用之后将出现荧光变化，如FRET显微镜所示。对现有技术提出了两点改进。首先，在培养细胞中设计和测试在单一载体(二合一)中包含两种融合蛋白的构建体。提出了几种可供选择的二合一载体系统设计，包括使用(1)内部酶切位点，(2)带有内部核糖体进入位点(IRES)的载体，或(3)具有两个串联的微型基因。这些载体产生的蛋白质将在单个细胞中进行蛋白质功能和能量转移测试，然后再用于整个组织。其次，将这两种融合蛋白在整个胚胎组织中进行转染和分析。这将需要对初级组织进行转基因。已经提出了几种替代方法，包括商业通透剂、电穿孔或病毒感染。将用流式细胞仪、GST下拉试验和Western blotts来评估转染率。Rho和ROCK之间的蛋白质-蛋白质相互作用以及所有适当的对照将成为FRET在全角膜上皮细胞中的第一个测试对。一旦程序被开发出来，探针的数量将扩大到研究Rho激活-信号通路中的其他上游和下游蛋白质，在未来的赠款应用中。这是将已建立的组织培养或单细胞技术转移到整个胚胎组织中。这些技术的发展将对未来眼睛发育的研究产生重大影响，因为蛋白质-蛋白质的直接相互作用可能是可视化的。这个项目的风险很高，因为“二合一”载体和整个组织的转基因都没有完成。

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.