OptoTFs: Genetically encoded light-activated transcription factors

描述（由申请人提供）：控制细胞、组织和活体动物中的神经元功能的能力对于理解特定蛋白质在神经功能和行为中的作用至关重要。由于光对内源性细胞功能的影响有限，因此光控制蛋白质功能已成为一种流行的方法。然而，除了微生物视蛋白外，很少有蛋白质在哺乳动物细胞中表达时可以受光调节。本应用程序描述了一种新型蛋白质的设计，构建，优化和应用，optotf，这是一种遗传编码的光激活转录因子。optotf将允许细胞、组织或动物中的基因转录以空间和时间可控的方式由光控制。本应用中的实验旨在优化光刺激后具有CREB活性的OptoTF的构建。光激活游离神经元培养和器官型切片培养中CREB活性的能力将得到优化。利用这种optoTF， CREB在树突形态发生中的作用也将被研究。本应用程序中的实验将开发一种全新的方法，利用光在空间和时间上操纵基因转录。此外，这里建立的方法很可能可推广到其他类型的蛋白质，从而有可能使光遗传学控制广泛的蛋白质用于许多应用。本应用程序描述了一种新型蛋白质的设计，构建，优化和应用，optotf，这是一种遗传编码的光激活转录因子。optotf将成为控制细胞、组织和活体动物基因表达的新工具。该工具的开发将使未来的研究能够确定基因转录在学习，记忆和成瘾中的作用。

DESCRIPTION (provided by applicant): The ability to control neuronal function in cells, tissues, and living animals is critical for understanding the role of specific proteins in neural function and behavior. Controlling protein function by light has emerged as a popular approach due to its limited effects on endogenous cellular function. However, other than microbial opsins, few proteins when expressed in mammalian cells can be regulated by light. This application describes the design, construction, optimization, and application of a new class of proteins, the optoTFs, which are genetically encoded, light-activated transcription factors. OptoTFs will allow gene transcription in cells, tissue, or animals to be controlled by light in a spatially and temporally controlled manner. Experiments in this application seek to optimize the construction of an OptoTF that has CREB activity following light stimulation. The ability of light to activate CREB activity in dissociated neuronal culture and in organotypic slice culture will be optimized. Using this optoTF, the role of CREB in dendrite morphogenesis will also be investigated. The experiments in this application will develop a fundamentally novel approach to use light to spatially and temporally manipulate gene transcription. Furthermore, the methods established here are likely to be generalizable to other types of proteins, thereby potentially enabling the optogenetic control of a wide range of proteins for numerous applications. This application describes the design, construction, optimization, and application of a new class of proteins, the optoTFs, which are genetically encoded, light-activated transcription factors. OptoTFs will serve as a novel tool for the control of gene expression in cells, tissues and living animals. The development of this tool will enable future studies to establish the role of gene transcription in learning, memory, and addiction.