Production of glycoprotein-deleted rabies viruses for monosynaptic tracing and high-level gene expression in neurons.
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.
Wickersham
Ian R
IR
Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. wickersham@mit.edu
Sullivan
Heather A
HA
Seung
H Sebastian
HS
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D017353:Gene Deletion; D015870:Gene Expression; D005814:Genes, Viral; D006801:Humans; D009474:Neurons / Q000821:virology*; D011820:Rabies virus / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity / Q000502:physiology*; D011995:Recombination, Genetic; D014461:Ultracentrifugation; D014759:Viral Envelope Proteins / Q000235:genetics*; D014776:Virus Cultivation / Q000379:methods*; D014779:Virus Replication / Q000235:genetics
D014759:Viral Envelope Proteins