Abstract

Genetic engineering by viral infection of single cells is useful to study complex systems such as the brain. However, available methods for infecting single cells have drawbacks that limit their applications. Here we describe 'virus stamping', in which viruses are reversibly bound to a delivery vehicle—a functionalized glass pipette tip or magnetic nanoparticles in a pipette—that is brought into physical contact with the target cell on a surface or in tissue, using mechanical or magnetic forces. Different single cells in the same tissue can be infected with different viruses and an individual cell can be simultaneously infected with different viruses. We use rabies, lenti, herpes simplex, and adeno-associated viruses to drive expression of fluorescent markers or a calcium indicator in target cells in cell culture, mouse retina, human brain organoid, and the brains of live mice. Virus stamping provides a versatile solution for targeted single-cell infection of diverse cell types, both in vitro and in vivo.

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Acknowledgements

We thank J. Gründemann for technical assistance setting up the brain slice procedure, J.M. Mateos, A. Kaech, and J. Doehner from the Zurich Center of Microscopy and Image Analysis (ZMB), U. Schwarz from Leica Mannheim, and T. Horn from the DBSSE imaging facility for helping with imaging and data preparation, M.J. Schnell for providing the BSR-VSV-RVG cell line, E.M. Callaway for providing the B7GG cell line, C.P. Patino Alvarez and A. Villemain for producing viruses, Helbling Technik Bern AG for technical assistance modeling the magnet used for shielded virus stamping, and the members of the Roska laboratory for technical assistance. The study was supported by a European Union grant (FP7/211800) to D.J.M.; a Human Frontier Science Program Long-Term Fellowship (LT000173/2013-L) and a Swiss National Science Foundation Ambizione Fellowship to S.T.; a European Molecular Biology Organization Long-Term Fellowship (506-2012) to D.M.M.; Swiss National Science Foundation grants (310030B_160225 to D.J.M. and 3100330B_163457 to B.R.), the National Center of Competence in Research Molecular Systems Engineering grant to D.J.M. and B.R.; European Research Council (669157, RETMUS), DARPA (HR0011-17-C-0038, Cortical Sight) grants to B.R., a Deutsche Forschungsgemeinschaft grant (SFB870) to K.K.C.

Author information

Author notes

    • Stuart Trenholm
    •  & Keisuke Yonehara

    Present addresses: Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada (S.T.) and The Danish Research Institute of Translational Neuroscience, Aarhus University, Aarhus, Denmark (K.Y.).

    • Rajib Schubert
    • , Stuart Trenholm
    •  & Kamill Balint

    These authors contributed equally to this work.

Affiliations

  1. Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland.

    • Rajib Schubert
    • , Manuel A Mohr
    • , David Martinez-Martin
    • , Gotthold Fläschner
    • , Richard Newton
    • , Aaron Ponti
    •  & Daniel J Müller
  2. Neural Circuit Laboratories, Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

    • Stuart Trenholm
    • , Kamill Balint
    • , Georg Kosche
    • , Cameron S Cowan
    • , Martin Munz
    • , Jacek Krol
    • , Brigitte Gross Scherf
    • , Keisuke Yonehara
    • , Adrian Wertz
    • , Daniel Hillier
    •  & Botond Roska
  3. University of Basel, Basel, Switzerland.

    • Kamill Balint
  4. Max von Pettenkofer-Institute & Gene Center, Faculty of Medicine, Ludwig-Maximilians-University Munich, Munich, Germany.

    • Alexander Ghanem
    •  & Karl-Klaus Conzelmann
  5. Department of Ophthalmology, University of Basel, Basel, Switzerland.

    • Botond Roska

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Contributions

Experiments were designed by R.S., S.T., K.B., G.K., G.F., D.J.M., and B.R. G-deleted rabies variants were made by A.G. and K.K.C. Cell cultures, immunohistochemistry, electron microscopy, and confocal microscopy were performed by R.S. with the exception of in vivo samples, which were processed by A.W., and organoid samples, which were processed by M.M. Viruses were prepared by R.S., K.B., M.A.M., R.N., and K.Y. Pipettes for unshielded virus stamping were prepared by D.M.M. Brain slice preparations, retinal preparations, unshielded virus stamping, and tissue cultures were performed by S.T. and K.B. The magnetic forces related to the magnet used for shielded stamping were measured and modeled by R.S. and C.S.C. Sequential multi-day single-cell infection experiments were performed by R.S. and C.S.C. Magnetic nanoparticle preparations and shielded stamping in cell culture were performed by R.S. In vivo nanoparticle optimization was performed by D.H. Shielded in vivo stamping was performed by S.T. and G.K. Organoids were grown by M.M., J.K., and B.G.S. Shielded organoid stamping was performed by G.K. and M.M. In vivo two-photon calcium imaging was performed by G.K. Computer reconstructions were performed by R.S., M.A.M., and A.P. Figures were made by R.S., S.T., M.M., and G.K. The paper was written by R.S., S.T., D.J.M., and B.R.

Competing interests

R.S., S.T., G.F., D.J.M. and B.R. applied for a patent related to viral stamping approach.

Corresponding authors

Correspondence to Daniel J Müller or Botond Roska.

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    Supplementary Note 1

    Modeling the electromagnet used for virus stamping and its interaction with the virus-coated nanoparticles

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DOI

https://doi.org/10.1038/nbt.4034