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Production of glycoprotein-deleted rabies viruses for monosynaptic tracing and high-level gene expression in neurons

Abstract

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.

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Figure 1: Flow diagram of the protocol.
Figure 2: Flow cytometry screen snapshot.
Figure 3: Isolated cluster of cells infected by spreading virus in transfection plate, 4 d after transfection.

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Acknowledgements

We thank Jennifer Wang and Jonathan Nassi for comments on the manuscript and Lynn Enquist, Rachael Neve, Sean Whelan and John Rose for helpful discussions. The genomic vector cSPBN was kindly provided by Dr Matthias Schnell, the vectors pTIT-N, pTIT-P, pTIT-G and pTIT-L were a generous gift from Dr Karl-Klaus Conzelmann and the plasmid pCAGGS-T7 was a generous gift from Dr Robert Lamb. Funding was provided by the Howard Hughes Medical Institute.

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I.R.W. and H.A.S. carried out all experiments; H.S.S. supervised all experiments.

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Correspondence to Ian R Wickersham.

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Wickersham, I., Sullivan, H. & Seung, H. Production of glycoprotein-deleted rabies viruses for monosynaptic tracing and high-level gene expression in neurons. Nat Protoc 5, 595–606 (2010). https://doi.org/10.1038/nprot.2009.248

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