Anterograde viral tracers are powerful and essential tools for dissecting the output targets of a brain region of interest. They have been developed from herpes simplex virus 1 (HSV-1) strain H129 (H129), and have been successfully applied to map diverse neural circuits. Initially, the anterograde polysynaptic tracer H129-G4 was used by many groups. We then developed the first monosynaptic tracer, H129-dTK-tdT, which was highly successful, yet improvements are needed. Now, by inserting another tdTomato expression cassette into the H129-dTK-tdT genome, we have created H129-dTK-T2, an updated version of H129-dTK-tdT that has improved labeling intensity. To help scientists produce and apply our H129-derived viral tracers, here we provide the protocol describing our detailed and standardized procedures. Commonly-encountered technical problems and their solutions are also discussed in detail. Broadly, the dissemination of this protocol will greatly support scientists to apply these viral tracers on a large scale.

顺行病毒示踪剂是解剖感兴趣的大脑区域的输出目标的强大且重要的工具。它们是从单纯疱疹病毒 1 (HSV-1) H129 株 (H129) 开发而来，并已成功应用于绘制不同的神经回路。最初，许多小组使用顺行多突触示踪剂 H129-G4。然后，我们开发了第一个单突触示踪剂 H129-dTK-tdT，该产品非常成功，但仍需要改进。现在，通过将另一个 tdTomato 表达盒插入 H129-dTK-tdT 基因组中，我们创建了 H129-dTK-T2，它是 H129-dTK-tdT 的更新版本，提高了标记强度。为了帮助科学家生产和应用我们的 H129 衍生病毒示踪剂，我们在这里提供了描述我们详细和标准化程序的协议。还详细讨论了常见的技术问题及其解决方案。从广义上讲，该协议的传播将极大地支持科学家大规模应用这些病毒示踪剂。