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Design and generation of recombinant rabies virus vectors

Nature Protocols volume 8, pages 1583–1601 (2013)Cite this article

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Abstract

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.

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Figure 1: Flowchart and timeline for G-deleted rabies virus production.
Figure 2: Rabies virus genome and G-deleted rabies virus vector.
Figure 3: Typical scheme for G-deleted rabies virus production.
Figure 4: Recovery of G-deleted rabies viruses from DNA plasmid.
Figure 5: Amplification of G-deleted rabies viruses in B7GG cells.

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Acknowledgements

We thank K.-K. Conzelmann, J.A.T. Young, I.R. Wickersham, T. Mori, J. Choi, N.R. Wall and A.H. Cetin for discussions, and B. Virgen, K.v. Bochmann and M. De La Parra for technical assistance. F.O. is thankful to N. Osakada for constant encouragement and support. We are grateful for the support from the US National Institutes of Health (grant nos. MH063912, NS069464 and EY022577 to E.M.C.), the Kavli Institute for Brain and Mind at the University of California San Diego (to E.M.C.), the Gatsby Charitable Foundation (to E.M.C.), the Japan Society for the Promotion of Science (to F.O.), the Kanae Foundation for the Promotion of Medical Science (to F.O.), the Uehara Memorial Foundation (to F.O.), the Naito Foundation (to F.O.) and the Pioneer Fund (to F.O.).

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  1. Systems Neurobiology Laboratories, The Salk Institute for Biological Studies, La Jolla, California, USA

    Fumitaka Osakada & Edward M Callaway

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F.O. designed and performed experiments, analyzed data and wrote the paper. E.M.C. supervised the project and provided feedback on the manuscript.

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Correspondence to Edward M Callaway.

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Osakada, F., Callaway, E. Design and generation of recombinant rabies virus vectors. Nat Protoc 8, 1583–1601 (2013). https://doi.org/10.1038/nprot.2013.094

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