Light-Induced Protein Expression

描述（由申请人提供）： 拟议的研究计划的总体目标是开发一种基因激活技术，可以应用于空间离散的组织亚位点的成年动物。这项技术的重要性在于，它应该提供一种直接的手段来检查特定组织微环境中基因表达的生物学后果。例如，乳腺和前列腺肿瘤的生长和进展取决于它们发育的微环境。然而，将特定蛋白质的活性（或其缺乏）与作为组织微环境的函数的致癌潜力相关联尚不可行。我们的策略基于埃文斯和他的同事开发的蜕皮激素驱动的基因表达系统，并由佩斯特尔和他的同事在阿尔伯特·爱因斯坦医学院进一步完善。我们已经制备了一种光激活形式的蜕皮激素，与点照明相结合，可以驱动多细胞环境中存在的任何所需细胞亚群中的蛋白质表达。我们将准备笼蜕皮类固醇，可以光化学释放使用双光子照明。这些光活化剂的基因活化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，后一种技术可以更深地穿透组织，并显著降低光毒性负荷。因此，它是唯一适用于整个动物系统。我们将在两种不同但常用的动物模型中评估光诱导蛋白表达的功效。第一个模型由在Condeelis组中常规使用的Fischer 344大鼠中异位表达的肿瘤组成。第二个模型是先前描述的在Pestell组中开发的三重转基因小鼠系。

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.