Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Nat Protoc. 2010 Mar;5(3):595-606. doi: 10.1038/nprot.2009.248. Epub 2010 Mar 4.
Recombinant rabies viruses rendered replication-deficient by the deletion of their envelope glycoprotein gene are useful tools for neuroscientists, permitting (1) extraordinarily high transgene expression levels within neurons, (2) retrograde infection of projection neurons through their axon terminals, (3) targeted infection of genetically specified neurons and (4) monosynaptic tracing of neuronal inputs. Here we present a detailed protocol for the production of high-titer and high-purity viral stocks, from initial generation of infectious virus from cDNA through amplification on complementing cell lines, pseudotyping if desired, purification by ultracentrifugation and titering. The procedure requires 3-4 weeks to complete.
缺失包膜糖蛋白基因而致复制缺陷的重组狂犬病病毒是神经科学家的有用工具，可实现（1）神经元内极高的转基因表达水平，（2）通过轴突末梢逆行感染投射神经元，（3）对特定遗传神经元的靶向感染，以及（4）神经元输入的单突触示踪。本文提供了一个详细的从 cDNA 最初生成感染性病毒到互补细胞系扩增、如有需要进行假型化、超速离心纯化和滴度测定的高滴度和高纯度病毒储备液的生产方案。该过程需要 3-4 周完成。
