Department of Circuits-Computations-Models, Max Planck Institute of Neurobiology, 82152 Martinsried, Germany.
G3 (Bethesda). 2022 Sep 30;12(10). doi: 10.1093/g3journal/jkac178.
The ability to drive expression of exogenous genes in different tissues and cell types, under the control of specific enhancers, has been crucial for discovery in biology. While many enhancers drive expression broadly, several genetic tools were developed to obtain access to isolated cell types. Studies of spatially organized neuropiles in the central nervous system of fruit flies have raised the need for a system that targets subsets of cells within a single neuronal type, a feat currently dependent on stochastic flip-out methods. To access the same cells within a given expression pattern consistently across fruit flies, we developed the light-gated expression system LOV-LexA. We combined the bacterial LexA transcription factor with the plant-derived light, oxygen, or voltage photosensitive domain and a fluorescent protein. Exposure to blue light uncages a nuclear localizing signal in the C-terminal of the light, oxygen, or voltage domain and leads to the translocation of LOV-LexA to the nucleus, with the subsequent initiation of transcription. LOV-LexA enables spatial and temporal control of expression of transgenes under LexAop sequences in larval fat body and pupal and adult neurons with blue light. The LOV-LexA tool is ready to use with GAL4 and Split-GAL4 drivers in its current form and constitutes another layer of intersectional genetics that provides light-controlled genetic access to specific cells across flies.
在不同组织和细胞类型中,通过特定增强子控制外源基因的表达能力,对于生物学的发现至关重要。虽然许多增强子广泛驱动表达,但已经开发了几种遗传工具来获取对分离细胞类型的访问权限。对果蝇中枢神经系统中空间组织神经叶的研究提出了一种需要针对单个神经元类型内的细胞亚群进行靶向的系统的需求,而这一壮举目前依赖于随机翻转方法。为了在给定的表达模式内一致地访问给定果蝇中的相同细胞,我们开发了光门控表达系统 LOV-LexA。我们将细菌 LexA 转录因子与源自植物的光、氧或电压光敏结构域和荧光蛋白结合在一起。暴露于蓝光会使光、氧或电压结构域的 C 末端的核定位信号去笼,并导致 LOV-LexA 易位到细胞核,随后启动转录。LOV-LexA 能够在幼虫脂肪体和蛹及成虫神经元中的 LexAop 序列下,对转基因进行时空控制表达。LOV-LexA 工具在其当前形式下已可与 GAL4 和 Split-GAL4 驱动子一起使用,并且构成了另一个交叉遗传层次,为特定细胞提供了跨果蝇的光控遗传访问。