Light-Induced Protein Expression

描述(由申请人提供)： 拟议研究计划的总体目标是开发一种可应用于成年动物空间离散组织亚基的基因激活技术。这项技术的重要性在于，它应该提供一种直接的手段，在特定组织微环境的背景下检查基因表达的生物学后果。例如，乳腺癌和前列腺癌的生长和发展取决于它们生长的微环境。然而，将特定蛋白质的活性(或缺乏活性)与致癌潜力作为组织微环境的函数相关联还不可行。我们的策略是基于由Evans和他的同事开发的蜕皮激素驱动的基因表达系统，并由Pestell和他的同事在阿尔伯特·爱因斯坦医学院进一步完善。我们已经准备了一种光激活形式的蜕皮激素，与斑点照明相结合，可以在多细胞环境中存在的任何所需细胞亚群中驱动蛋白质表达。我们将准备笼子里的蜕皮激素，可以使用双光子照明来光化学释放。这些光激活剂的基因激活特性将在无细胞和基于细胞的模型系统中以及在存在或不存在双光子照明的情况下进行评估。与单光子照射相比，后一种技术提供了更深的组织穿透和显著减少的光毒性负荷。因此，它是唯一适用于整个动物系统的。我们将在两个不同但常用的动物模型中评估光诱导蛋白质表达的有效性。第一个模型由Condeelis组常规使用的Fischer 344大鼠的异位表达肿瘤组成。第二个模型是之前描述的由Pestell小组开发的三重转基因小鼠品系。

DESCRIPTION (provided by applicant): The overall goal of the proposed research program is to develop a gene activation technology that can be applied to spatially discrete tissue subsites in the adult animal. The importance of this technology is that it should furnish a direct means to examine the biological consequences of gene expression within the context of specific tissue microenvironments. For example, the growth and progression of breast and prostate tumors are dependent upon the microenvironment in which they develop. However, it is not yet feasible to correlate the activity (or lack thereof) of a particular protein with oncogenic potential as a function of tissue microenvironment. Our strategy is based upon the ecdysteroid-driven gene expression system developed by Evans and his colleagues and further refined at the Albert Einstein College of Medicine by Pestell and his coworkers. We have prepared a light-activatable form of ecdysone that, in combination with spot illumination, can drive protein expression in any desired subset of cells present in a multicellular environment. We wilt prepare caged ecdysteroids that can be photochemically unleashed using two-photon illumination. The gene activation properties of these light-activatable agents will be evaluated both in cell-free and cell-based model systems and in the presence or absence of two-photon illumination. The latter technology furnishes deeper tissue penetration and a significantly reduced phototoxic load compared to single photon irradiation. Consequently, it is uniquely applicable to whole animal systems. We will evaluate the efficacy of light-induced protein expression in two distinct, but commonly employed, animal models. The first model consists of ectopically expressed tumors in Fischer 344 rats routinely employed in the Condeelis group. The second model is a previously described triply transgenic mouse line developed in the Pestell group.