Systems Neurobiology Laboratories, The Salk Institute for Biological Studies, La Jolla, California, USA.
Nat Protoc. 2013 Aug;8(8):1583-601. doi: 10.1038/nprot.2013.094. Epub 2013 Jul 25.
Rabies viruses, negative-strand RNA viruses, infect neurons through axon terminals and spread trans-synaptically in a retrograde direction between neurons. Rabies viruses whose glycoprotein (G) gene is deleted from the genome cannot spread across synapses. Complementation of G in trans, however, enables trans-synaptic spreading of G-deleted rabies viruses to directly connected, presynaptic neurons. Recombinant rabies viruses can encode genes of interest for labeling cells, controlling gene expression and monitoring or manipulating neural activity. Cre-dependent or bridge protein-mediated transduction and single-cell electroporation via the EnvA-TVA or EnvB-TVB (envelope glycoprotein and its specific receptor for avian sarcoma leukosis virus subgroup A or B) system allow cell type-specific or single cell-specific targeting. These rabies virus-based approaches permit the linking of connectivity to cell morphology and circuit function for particular cell types or single cells. Here we describe methods for construction of rabies viral vectors, recovery of G-deleted rabies viruses from cDNA, amplification of the viruses, pseudotyping them with EnvA or EnvB and concentration and titration of the viruses. The entire protocol takes 6-8 weeks.
狂犬病病毒是负链 RNA 病毒，通过轴突末梢感染神经元，并在神经元之间沿逆行的突触间隙进行传播。缺失糖蛋白 (G) 基因的狂犬病病毒不能跨越突触传播。然而，通过反式互补 G，可使 G 缺失的狂犬病病毒在直接连接的、突触前神经元之间进行顺行传播。重组狂犬病病毒可编码感兴趣的基因，用于标记细胞、控制基因表达以及监测或操纵神经活动。依赖 Cre 或桥接蛋白的转导以及通过 EnvA-TVA 或 EnvB-TVB（包膜糖蛋白及其特定的禽肉瘤白血病病毒亚群 A 或 B 的受体）系统的单细胞电穿孔允许针对特定细胞类型或单细胞进行靶向。这些基于狂犬病病毒的方法允许将连接性与特定细胞类型或单个细胞的细胞形态和回路功能联系起来。本文描述了狂犬病病毒载体的构建、从 cDNA 中回收 G 缺失的狂犬病病毒、病毒的扩增、用 EnvA 或 EnvB 对其进行假型化以及病毒的浓缩和滴定的方法。整个方案需要 6-8 周时间。
