Chen Xianjun, Wang Xue, Du Zengmin, Ma Zhengcai, Yang Yi
Synthetic Biology and Biotechnology Laboratory, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science and Technology, Shanghai, China.
Curr Protoc Chem Biol. 2013;5(2):111-29. doi: 10.1002/9780470559277.ch120267.
A light-switchable transgene system could be a powerful optogenetic tool for the precise manipulation of spatiotemporal gene expression in multicellular organisms. We have developed the LightOn system, which consists of a single chimeric protein (GAVPO) that can homodimerize and bind to promoters upon exposure to blue light, activating transcription of a target gene. This article describes protocols for precise control of gene expression in mammalian cells and mice using the LightOn system. These protocols can be carried out in an ordinary laboratory, as both liposome-mediated transfection and hydrodynamic tail vein injection are routine methods that can easily transfer the LightOn system to mammalian cells and mouse liver, respectively. The illumination equipment can also be easily obtained. The LightOn system can provide a robust, convenient means to control the expression of a gene of interest, with unprecedented temporal and spatial accuracy in manipulating an extremely broad range of biological processes.
一种光开关转基因系统可能是一种强大的光遗传学工具,用于在多细胞生物中精确操纵时空基因表达。我们开发了LightOn系统,它由一种单一的嵌合蛋白(GAVPO)组成,该蛋白在暴露于蓝光时可以同源二聚化并与启动子结合,从而激活靶基因的转录。本文描述了使用LightOn系统精确控制哺乳动物细胞和小鼠基因表达的实验方案。这些方案可以在普通实验室中进行,因为脂质体介导的转染和流体动力学尾静脉注射都是常规方法,它们可以分别轻松地将LightOn系统转移到哺乳动物细胞和小鼠肝脏中。照明设备也很容易获得。LightOn系统可以提供一种强大、便捷的手段来控制感兴趣基因的表达,在操纵极其广泛的生物过程中具有前所未有的时间和空间精度。