Uchigashima Motokazu, Mikuni Takayasu
Department of Cellular Neuropathology, Brain Research Institute, Niigata University, 1-757, Asahimachi-dori, Niigata, 951-8585, Japan.
International Research Center for Neurointelligence (WPI-IRCN), The University of Tokyo, Tokyo, Japan.
Anat Sci Int. 2025 Jul 14. doi: 10.1007/s12565-025-00866-x.
High-precision mapping of endogenous proteins is essential for understanding the molecular mechanism underlying neuronal functions in the brain. The SLENDR (single-cell labeling of endogenous proteins by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated homology-directed repair) technique provides single-cell endogenous protein labeling with genetically encoded tags within the mammalian brain through precise genome editing via homology-directed repair (HDR). This technique is based on the introduction of HDR-mediated genome editing into neuronal progenitors in embryonic brains by in utero electroporation. Subsequent histological analyses enable high-resolution interrogation of the subcellular distribution of endogenous proteins within a single neuron using conventional fluorescent microscopy. Here, we describe a step-by-step protocol for the SLENDR technique to label endogenous proteins with genetically encoded tags in single pyramidal cells of the mouse primary somatosensory cortex. This protocol would be helpful to visualize the molecular organization underlying biological processes at single-neuron levels in the brain, such as signal processing from synaptic inputs to neuronal outputs across different scales.
内源性蛋白质的高精度定位对于理解大脑中神经元功能的分子机制至关重要。SLENDR（通过成簇规律间隔短回文重复序列（CRISPR）-Cas9介导的同源定向修复对内源性蛋白质进行单细胞标记）技术通过同源定向修复（HDR）进行精确的基因组编辑，在哺乳动物大脑中为内源性蛋白质提供带有基因编码标签的单细胞标记。该技术基于通过子宫内电穿孔将HDR介导的基因组编辑引入胚胎大脑中的神经元祖细胞。随后的组织学分析能够使用传统荧光显微镜对单个神经元内源性蛋白质的亚细胞分布进行高分辨率研究。在这里，我们描述了一种用于SLENDR技术的逐步方案，以在小鼠初级体感皮层的单个锥体细胞中用基因编码标签标记内源性蛋白质。该方案将有助于在单神经元水平上可视化大脑中生物过程的分子组织，例如从突触输入到不同尺度上神经元输出的信号处理。
