Takahashi Megumu, Mizuno Hidenobu, Koike Masato, Iwasato Takuji, Hioki Hiroyuki
Center for Genomic and Regenerative Medicine, Juntendo University Graduate School of Medicine, Tokyo, Japan.
Department of Neuroanatomy, Juntendo University Graduate School of Medicine, Tokyo, Japan.
Anat Sci Int. 2025 Aug 8. doi: 10.1007/s12565-025-00867-w.
Adeno-associated virus (AAV) vectors are widely used to label individual neurons by expressing fluorescent proteins. To analyze neuronal morphology in detail, it is important to achieve sparse and strong expression of fluorescent proteins for clearly visualizing cell bodies, dendrites, and axons. However, injections of high-titer AAV vectors often result in excessive cell labeling, while injecting low-titer AAV vectors reduces signal intensity. Here, we provide a detailed, step-by-step protocol for achieving sparse and bright neuronal labeling using dual AAV vectors with Cre recombination, based on the Supernova system. We prepared two types of AAV vectors: one is a driver vector (AAV2/1-SYN-iCre-BGHpA) that expresses Cre recombinase under a neuron-specific promoter, and the other is a reporter vector (AAV2/1-SynTetOff FLEX-GFP) that encodes GFP with a flip-excision switch (FLEX) and the Tet-Off system. When a mixture of a serially diluted reporter vector and a high-titer driver vector was stereotaxically injected into the mouse brain, the number of labeled neurons and the fluorescence intensity of GFP decreased in a dose-dependent manner. Conversely, diluting the driver vector decreased the number of GFP-expressing cells while the fluorescence intensity of GFP remained high. Furthermore, this approach proved effective for systemic delivery via retro-orbital injection of AAV2/B10 vectors. This protocol includes comprehensive reagent lists, stepwise injection procedures, and troubleshooting tips, allowing researchers to reproducibly achieve sparse and bright labeling through both stereotaxic and retro-orbital injections.
腺相关病毒（AAV）载体被广泛用于通过表达荧光蛋白来标记单个神经元。为了详细分析神经元形态，实现荧光蛋白的稀疏且强表达以清晰可视化细胞体、树突和轴突非常重要。然而，注射高滴度AAV载体常常导致过度的细胞标记，而注射低滴度AAV载体则会降低信号强度。在此，我们基于超新星系统提供了一份详细的、逐步的方案，用于使用具有Cre重组的双AAV载体实现稀疏且明亮的神经元标记。我们制备了两种类型的AAV载体：一种是驱动载体（AAV2/1-SYN-iCre-BGHpA），其在神经元特异性启动子下表达Cre重组酶；另一种是报告载体（AAV2/1-SynTetOff FLEX-GFP），其通过翻转切除开关（FLEX）和Tet-Off系统编码绿色荧光蛋白（GFP）。当将系列稀释的报告载体和高滴度驱动载体的混合物立体定向注射到小鼠脑中时，标记神经元的数量和GFP的荧光强度呈剂量依赖性降低。相反，稀释驱动载体可减少表达GFP的细胞数量，而GFP的荧光强度仍保持较高。此外，该方法经证明通过眶后注射AAV2/B10载体进行全身递送是有效的。该方案包括全面的试剂清单、逐步注射程序和故障排除提示，使研究人员能够通过立体定向注射和眶后注射可重复地实现稀疏且明亮的标记。
