Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Design and generation of recombinant rabies virus vectors

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

References

  1. Luo, L., Callaway, E.M. & Svoboda, K. Genetic dissection of neural circuits. Neuron 57, 634–660 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Arenkiel, B.R. & Ehlers, M.D. Molecular genetics and imaging technologies for circuit-based neuroanatomy. Nature 461, 900–907 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Scanziani, M. & Hausser, M. Electrophysiology in the age of light. Nature 461, 930–939 (2009).

    Article  CAS  PubMed  Google Scholar 

  4. Yizhar, O., Fenno, L.E., Davidson, T.J., Mogri, M. & Deisseroth, K. Optogenetics in neural systems. Neuron 71, 9–34 (2011).

    Article  CAS  PubMed  Google Scholar 

  5. Carandini, M. & Heeger, D.J. Normalization as a canonical neural computation. Nat. Rev. Neurosci. 13, 51–62 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Wickersham, I.R., Finke, S., Conzelmann, K.K. & Callaway, E.M. Retrograde neuronal tracing with a deletion-mutant rabies virus. Nat. Methods 4, 47–49 (2007).

    Article  CAS  PubMed  Google Scholar 

  7. Wickersham, I.R. et al. Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons. Neuron 53, 639–647 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Larsen, D.D., Wickersham, I.R. & Callaway, E.M. Retrograde tracing with recombinant rabies virus reveals correlations between projection targets and dendritic architecture in layer 5 of mouse barrel cortex. Front Neural Circuits 1, 5 (2007).

    PubMed  Google Scholar 

  9. Nassi, J.J. & Callaway, E.M. Specialized circuits from primary visual cortex to V2 and area MT. Neuron 55, 799–808 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Choi, J., Young, J.A. & Callaway, E.M. Selective viral vector transduction of ErbB4-expressing cortical interneurons in vivo with a viral receptor-ligand bridge protein. Proc. Natl Acad. Sci. USA 107, 16703–16708 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Wall, N.R., Wickersham, I.R., Cetin, A., De La Parra, M. & Callaway, E.M. Monosynaptic circuit tracing in vivo through Cre-dependent targeting and complementation of modified rabies virus. Proc. Natl Acad. Sci. USA 107, 21848–21853 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Marshel, J.H., Mori, T., Nielsen, K.J. & Callaway, E.M. Targeting single neuronal networks for gene expression and cell labeling in vivo. Neuron 67, 562–574 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Osakada, F. et al. New rabies virus variants for monitoring and manipulating activity and gene expression in defined neural circuits. Neuron 71, 617–631 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Choi, J. & Callaway, E.M. Monosynaptic inputs to ErbB4-expressing inhibitory neurons in mouse primary somatosensory cortex. J. Comp. Neurol. 519, 3402–3414 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Nhan, H.L. & Callaway, E.M. Morphology of superior colliculus- and middle temporal area-projecting neurons in primate primary visual cortex. J. Comp. Neurol. 520, 52–80 (2012).

    Article  PubMed  Google Scholar 

  16. Stepien, A.E., Tripodi, M. & Arber, S. Monosynaptic rabies virus reveals premotor network organization and synaptic specificity of cholinergic partition cells. Neuron 68, 456–472 (2010).

    Article  CAS  PubMed  Google Scholar 

  17. Haubensak, W. et al. Genetic dissection of an amygdala microcircuit that gates conditioned fear. Nature 468, 270–276 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Yonehara, K. et al. Spatially asymmetric reorganization of inhibition establishes a motion-sensitive circuit. Nature 469, 407–410 (2011).

    Article  CAS  PubMed  Google Scholar 

  19. Miyamichi, K. et al. Cortical representations of olfactory input by trans-synaptic tracing. Nature 472, 191–196 (2011).

    Article  CAS  PubMed  Google Scholar 

  20. Rancz, E.A. et al. Transfection via whole-cell recording in vivo: bridging single-cell physiology, genetics and connectomics. Nat. Neurosci. 14, 527–532 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Tripodi, M., Stepien, A.E. & Arber, S. Motor antagonism exposed by spatial segregation and timing of neurogenesis. Nature 479, 61–66 (2011).

    Article  CAS  PubMed  Google Scholar 

  22. Kiritani, T., Wickersham, I.R., Seung, H.S. & Shepherd, G.M. Hierarchical connectivity and connection-specific dynamics in the corticospinal-corticostriatal microcircuit in mouse motor cortex. J. Neurosci. 32, 4992–5001 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Watabe-Uchida, M., Zhu, L., Ogawa, S.K., Vamanrao, A. & Uchida, N. Whole-brain mapping of direct inputs to midbrain dopamine neurons. Neuron 74, 858–873 (2012).

    Article  CAS  PubMed  Google Scholar 

  24. Vivar, C. et al. Monosynaptic inputs to new neurons in the dentate gyrus. Nat. Commun. 3, 1107 (2012).

    Article  CAS  PubMed  Google Scholar 

  25. Takatoh, J. et al. New modules are added to vibrissal premotor circuitry with the emergence of exploratory whisking. Neuron 77, 346–360 (2012).

    Article  CAS  Google Scholar 

  26. Garcia, I., Huang, L., Ung, K. & Arenkiel, B.R. Tracing synaptic connectivity onto embryonic stem cell-derived neurons. Stem Cells 30, 2140–2151 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Lammel, S. et al. Input-specific control of reward and aversion in the ventral tegmental area. Nature 491, 212–217 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Yin, L. et al. Imaging light responses of retinal ganglion cells in the living mouse eye. J. Neurophysiol. 109, 2415–2421 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Li, Y. et al. Molecular layer perforant path-associated cells contribute to feed-forward inhibition in the adult dentate gyrus. Proc. Natl Acad. Sci. USA 110, 9106–9111 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Ugolini, G. Specificity of rabies virus as a transneuronal tracer of motor networks: transfer from hypoglossal motoneurons to connected second-order and higher order central nervous system cell groups. J. Comp. Neurol. 356, 457–480 (1995).

    Article  CAS  PubMed  Google Scholar 

  31. Ugolini, G. Advances in viral transneuronal tracing. J. Neurosci. Methods 194, 2–20 (2010).

    Article  PubMed  Google Scholar 

  32. Callaway, E.M. Transneuronal circuit tracing with neurotropic viruses. Curr. Opin. Neurobiol. 18, 617–623 (2008).

    Article  CAS  PubMed  Google Scholar 

  33. Mebatsion, T., Konig, M. & Conzelmann, K.K. Budding of rabies virus particles in the absence of the spike glycoprotein. Cell 84, 941–951 (1996).

    Article  CAS  PubMed  Google Scholar 

  34. Etessami, R. et al. Spread and pathogenic characteristics of a G-deficient rabies virus recombinant: an in vitro and in vivo study. J. Gen. Virol. 81, 2147–2153 (2000).

    Article  CAS  PubMed  Google Scholar 

  35. Weible, A.P. et al. Transgenic targeting of recombinant rabies virus reveals monosynaptic connectivity of specific neurons. J. Neurosci. 30, 16509–16513 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Tian, L. et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat. Methods 6, 875–881 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Mank, M. et al. A genetically encoded calcium indicator for chronic in vivo two-photon imaging. Nat. Methods 5, 805–811 (2008).

    Article  CAS  PubMed  Google Scholar 

  38. Ohkura, M., Sasaki, T., Kobayashi, C., Ikegaya, Y. & Nakai, J. An improved genetically encoded red fluorescent Ca2+ indicator for detecting optically evoked action potentials. PLoS ONE 7, e39933 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Akemann, W., Mutoh, H., Perron, A., Rossier, J. & Knopfel, T. Imaging brain electric signals with genetically targeted voltage-sensitive fluorescent proteins. Nat. Methods 7, 643–649 (2010).

    Article  CAS  PubMed  Google Scholar 

  40. Kralj, J.M., Douglass, A.D., Hochbaum, D.R., Maclaurin, D. & Cohen, A.E. Optical recording of action potentials in mammalian neurons using a microbial rhodopsin. Nat. Methods 9, 90–95 (2012).

    Article  CAS  Google Scholar 

  41. Marvin, J.S. et al. An optimized fluorescent probe for visualizing glutamate neurotransmission. Nat. Methods 10, 162–170 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Lechner, H.A., Lein, E.S. & Callaway, E.M. A genetic method for selective and quickly reversible silencing of mammalian neurons. J. Neurosci. 22, 5287–5290 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Tan, E.M. et al. Selective and quickly reversible inactivation of mammalian neurons in vivo using the Drosophila allatostatin receptor. Neuron 51, 157–170 (2006).

    Article  CAS  PubMed  Google Scholar 

  44. Armbruster, B.N., Li, X., Pausch, M.H., Herlitze, S. & Roth, B.L. Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proc. Natl Acad. Sci. USA 104, 5163–5168 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Magnus, C.J. et al. Chemical and genetic engineering of selective ion channel-ligand interactions. Science 333, 1292–1296 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Boyden, E.S., Zhang, F., Bamberg, E., Nagel, G. & Deisseroth, K. Millisecond-timescale, genetically targeted optical control of neural activity. Nat. Neurosci. 8, 1263–1268 (2005).

    Article  CAS  PubMed  Google Scholar 

  47. Gradinaru, V. et al. Molecular and cellular approaches for diversifying and extending optogenetics. Cell 141, 154–165 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Chow, B.Y. et al. High-performance genetically targetable optical neural silencing by light-driven proton pumps. Nature 463, 98–102 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Petreanu, L., Huber, D., Sobczyk, A. & Svoboda, K. Channelrhodopsin-2–assisted circuit mapping of long-range callosal projections. Nat. Neurosci. 10, 663–668 (2007).

    Article  CAS  PubMed  Google Scholar 

  50. Lee, J.H. et al. Global and local fMRI signals driven by neurons defined optogenetically by type and wiring. Nature 465, 788–792 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Barnard, R.J., Elleder, D. & Young, J.A. Avian sarcoma and leukosis virus-receptor interactions: from classical genetics to novel insights into virus-cell membrane fusion. Virology 344, 25–29 (2006).

    Article  CAS  PubMed  Google Scholar 

  52. Seidler, B. et al. A Cre-loxP-based mouse model for conditional somatic gene expression and knockdown in vivo by using avian retroviral vectors. Proc. Natl Acad. Sci. USA 105, 10137–10142 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  53. Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010).

    Article  CAS  PubMed  Google Scholar 

  54. Taniguchi, H. et al. A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex. Neuron 71, 995–1013 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Madisen, L. et al. A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing. Nat. Neurosci. 15, 793–802 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Schnell, M.J., McGettigan, J.P., Wirblich, C. & Papaneri, A. The cell biology of rabies virus: using stealth to reach the brain. Nat. Rev. Microbiol. 8, 51–61 (2010).

    Article  CAS  PubMed  Google Scholar 

  57. Finke, S., Mueller-Waldeck, R. & Conzelmann, K.K. Rabies virus matrix protein regulates the balance of virus transcription and replication. J. Gen. Virol. 84, 1613–1621 (2003).

    Article  CAS  PubMed  Google Scholar 

  58. Hacein-Bey-Abina, S. et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302, 415–419 (2003).

    Article  CAS  PubMed  Google Scholar 

  59. Gonzalez, F., Boue, S. & Izpisua Belmonte, J.C. Methods for making induced pluripotent stem cells: reprogramming a la carte. Nat. Rev. Genet. 12, 231–242 (2011).

    Article  CAS  PubMed  Google Scholar 

  60. Schnell, M.J., Mebatsion, T. & Conzelmann, K.K. Infectious rabies viruses from cloned cDNA. EMBO J. 13, 4195–4203 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Wickersham, I.R., Sullivan, H.A. & Seung, H.S. Production of glycoprotein-deleted rabies viruses for monosynaptic tracing and high-level gene expression in neurons. Nat. Protoc. 5, 595–606 (2010).

    Article  CAS  PubMed  Google Scholar 

  62. Conzelmann, K.K., Cox, J.H., Schneider, L.G. & Thiel, H.J. Molecular cloning and complete nucleotide sequence of the attenuated rabies virus SAD B19. Virology 175, 485–499 (1990).

    Article  CAS  PubMed  Google Scholar 

  63. Schnell, M.J. et al. Recombinant rabies virus as potential live-viral vaccines for HIV-1. Proc. Natl Acad. Sci. USA 97, 3544–3549 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Mebatsion, T. & Conzelmann, K.K. Specific infection of CD4+ target cells by recombinant rabies virus pseudotypes carrying the HIV-1 envelope spike protein. Proc. Natl Acad. Sci. USA 93, 11366–11370 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Kato, S. et al. Efficient gene transfer via retrograde transport in rodent and primate brains using a human immunodeficiency virus type 1-based vector pseudotyped with rabies virus glycoprotein. Hum. Gene Ther. 18, 1141–1151 (2007).

    Article  CAS  PubMed  Google Scholar 

  66. Mazarakis, N.D. et al. Rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral delivery. Hum. Mol. Genet. 10, 2109–2121 (2001).

    Article  CAS  PubMed  Google Scholar 

  67. Lafon, M. Rabies virus receptors. J. Neurovirol. 11, 82–87 (2005).

    Article  CAS  PubMed  Google Scholar 

  68. Green, M.R. & Sambrook, J. Molecular Cloning: A Laboratory Manual (Fourth ed.) (Cold Spring Harbor Laboratory Press, 2012).

  69. Ryan, M.D. & Drew, J. Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein. EMBO J. 13, 928–933 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Vagner, S., Galy, B. & Pyronnet, S. Irresistible IRES. Attracting the translation machinery to internal ribosome entry sites. EMBO Rep. 2, 893–898 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Hasegawa, K., Cowan, A.B., Nakatsuji, N. & Suemori, H. Efficient multicistronic expression of a transgene in human embryonic stem cells. Stem Cells 25, 1707–1712 (2007).

    Article  CAS  PubMed  Google Scholar 

  72. Mizuguchi, H., Xu, Z., Ishii-Watabe, A., Uchida, E. & Hayakawa, T. IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector. Mol. Ther. 1, 376–382 (2000).

    Article  CAS  PubMed  Google Scholar 

  73. Le Mercier, P., Jacob, Y., Tanner, K. & Tordo, N. A novel expression cassette of lyssavirus shows that the distantly related Mokola virus can rescue a defective rabies virus genome. J. Virol. 76, 2024–2027 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Symons, R.H. Small catalytic RNAs. Annu. Rev. Biochem. 61, 641–671 (1992).

    Article  CAS  PubMed  Google Scholar 

  75. Gaudin, Y., Tuffereau, C., Segretain, D., Knossow, M. & Flamand, A. Reversible conformational changes and fusion activity of rabies virus glycoprotein. J. Virol. 65, 4853–4859 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  76. Gaudin, Y., Ruigrok, R.W., Knossow, M. & Flamand, A. Low-pH conformational changes of rabies virus glycoprotein and their role in membrane fusion. J. Virol. 67, 1365–1372 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

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.).

Author information

Authors and Affiliations

Authors

Contributions

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

Corresponding author

Correspondence to Edward M Callaway.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2013.094

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing