Department of Chemistry, University of California, Berkeley, California 94720, United States.
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, United States.
ACS Chem Neurosci. 2023 Mar 1;14(5):909-916. doi: 10.1021/acschemneuro.2c00745. Epub 2023 Feb 17.
Visualizing neuronal anatomy often requires labor-intensive immunohistochemistry on fixed and dissected brains. To facilitate rapid anatomical staining in live brains, we used genetically targeted membrane tethers that covalently link fluorescent dyes for in vivo neuronal labeling. We generated a series of extracellularly trafficked small-molecule tethering proteins, HaloTag-CD4 (Kirk et al. , , 754027) and SNAP-CD4, which directly label transgene-expressing cells with commercially available ligand-substituted fluorescent dyes. We created stable transgenic reporter lines, which express extracellular HaloTag-CD4 and SNAP-CD4 with LexA and Gal4 drivers. Expressing these enzymes in live brains, we labeled the expression patterns of various Gal4 driver lines recapitulating histological staining in live-brain tissues. Pan-neural expression of SNAP-CD4 enabled the registration of live brains to an existing template for anatomical comparisons. We predict that these extracellular platforms will not only become a valuable complement to existing anatomical methods but will also prove useful for future genetic targeting of other small-molecule probes, drugs, and actuators.
可视化神经元结构通常需要对固定和解剖的大脑进行费力的免疫组织化学处理。为了在活体大脑中实现快速的解剖染色，我们使用了基因靶向的膜系绳，将荧光染料共价连接起来，用于活体神经元标记。我们生成了一系列细胞外转运的小分子系绳蛋白，HaloTag-CD4（Kirk 等人，，754027）和 SNAP-CD4，它们可以直接用商业上可用的配体取代的荧光染料标记转基因表达细胞。我们创建了稳定的转基因报告系，这些系表达 LexA 和 Gal4 驱动子的细胞外 HaloTag-CD4 和 SNAP-CD4。在活体大脑中表达这些酶，我们标记了各种 Gal4 驱动子系的表达模式，重现了活体脑组织中的组织学染色。SNAP-CD4 的全神经元表达使活体大脑能够与现有的模板进行注册，以便进行解剖比较。我们预测，这些细胞外平台不仅将成为现有解剖方法的有价值的补充，而且对于未来对其他小分子探针、药物和执行器的遗传靶向也将非常有用。
