Wang Wenjing, Wildes Craig P, Pattarabanjird Tanyaporn, Sanchez Mateo I, Glober Gordon F, Matthews Gillian A, Tye Kay M, Ting Alice Y
Departments of Genetics, Biology, and Chemistry, Stanford University, Stanford, California, USA.
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Nat Biotechnol. 2017 Sep;35(9):864-871. doi: 10.1038/nbt.3909. Epub 2017 Jun 26.
Activity remodels neurons, altering their molecular, structural, and electrical characteristics. To enable the selective characterization and manipulation of these neurons, we present FLARE, an engineered transcription factor that drives expression of fluorescent proteins, opsins, and other genetically encoded tools only in the subset of neurons that experienced activity during a user-defined time window. FLARE senses the coincidence of elevated cytosolic calcium and externally applied blue light, which together produce translocation of a membrane-anchored transcription factor to the nucleus to drive expression of any transgene. In cultured rat neurons, FLARE gives a light-to-dark signal ratio of 120 and a high- to low-calcium signal ratio of 10 after 10 min of stimulation. Opsin expression permitted functional manipulation of FLARE-marked neurons. In adult mice, FLARE also gave light- and motor-activity-dependent transcription in the cortex. Due to its modular design, minute-scale temporal resolution, and minimal dark-state leak, FLARE should be useful for the study of activity-dependent processes in neurons and other cells that signal with calcium.
活动重塑神经元，改变其分子、结构和电学特性。为了能够对这些神经元进行选择性表征和操控，我们展示了FLARE，一种经过工程改造的转录因子，它仅在用户定义的时间窗口内经历过活动的神经元子集中驱动荧光蛋白、视蛋白和其他基因编码工具的表达。FLARE感知胞质钙升高与外部施加蓝光的同时发生，二者共同促使膜锚定转录因子转位至细胞核，从而驱动任何转基因的表达。在培养的大鼠神经元中，刺激10分钟后，FLARE的光暗信号比为120，高钙与低钙信号比为10。视蛋白的表达允许对FLARE标记的神经元进行功能操控。在成年小鼠中，FLARE在皮层中也呈现出依赖光和运动活动的转录。由于其模块化设计、分钟级的时间分辨率以及最小的暗态泄漏，FLARE应有助于研究神经元和其他以钙信号传导的细胞中依赖活动的过程。
