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<p>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.</p>

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A detailed protocol for sparse and bright labeling of neurons via dual adeno-associated virus vectors with Cre recombination

  • 期刊论文

  • 发表日期：

    2025年8月8日

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  • Megumu Takahashi,
  • Hidenobu Mizuno,
  • Masato Koike,
  • Takuji Iwasato,
  • Hiroyuki Hioki

摘要

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.