Abstract
To understand and control complex tissues, the ability to genetically manipulate single cells is required. However, current delivery methods for the genetic engineering of single cells, including viral transduction, suffer from limitations that restrict their application. Here we present a protocol that describes a versatile technique that can be used for the targeted viral infection of single cells or small groups of cells in any tissue that is optically accessible. First, cells of interest are selected using optical microscopy. Second, a micropipette—loaded with magnetic nanoparticles to which viral particles are bound—is brought into proximity of the cell of interest, and a magnetic field is applied to guide the viral nanoparticles into cellular contact, leading to transduction. The protocol, exemplified here by stamping cultured neurons with adeno-associated viruses (AAVs), is completed in a few minutes and allows stable transgene expression within a few days, at success rates that approach 80%. We outline how this strategy is applied to single-cell infection in complex tissues, and is feasible both in organoids and in vivo.
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The data shown in this protocol are available upon reasonable request.
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Acknowledgements
We thank P. Buchmann and P. Argast for building our magnet Supplementary Figure 1. The study was supported by an European Union grant (FP7/211800 to D.J.M.), a Canada Research Chair (Tier II to S.T.), Swiss National Science Foundation grants (310030B_160225 to D.J.M. and 3100330B_163457 to B.R.), the National Center of Competence in Research (NCCR) Molecular Systems Engineering (to D.J.M. and B.R.), the European Research Council (669157, RETMUS to B.R.) and a DARPA grant (HR0011-17-C-0038, Cortical Sight to B.R.).
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All authors discussed the protocol and contributed to the writing of the protocol. R.S., S.H. and S.T. designed and optimized the experiments described. Figures and videos were created by R.S., S.H. and S.T.
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R.S., S.T., D.J.M. and B.R. applied for a patent related to the virus-stamping approach. The remaining author declares no competing interests.
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Key references using this protocol
Schubert, R. et al. Nat. Biotechnol. 36, 81–88 (2018): https://doi.org/10.1038/nbt.4034
Wertz, A. et al. Science 349, 70–74 (2015): https://doi.org/10.1126/science.aab1687
Alsteens, D. et al. Nat. Nanotechnol. 12, 177–183 (2017): https://doi.org/10.1038/nnano.2016.228
Integrated supplementary information
Supplementary Fig. 1 Schematic of setup used for shielded virus stamping for experiments.
To maintain health of cells during the stamping procedure and allowing for glass capillary (pipette), optical and magnet access we built a custom chamber made of two concentric compartments31. The inner compartment is a glass-bottomed dish in which cells are cultured at 37 °C. The surrounding compartment is continuously filled via an inlet with humidified (<98% relative humidity) synthetic air (80% N2 and 20% O2) supplemented with 5% CO2. The top of the chamber is left open to access the cells by the glass capillary pipette and objective (top-down dipping objective). A 35 mm diameter opening of the outer chamber (chamber lid) allows optical access from the top. The angle between pipette and glass-bottom dish, which is usually ~ 45° can be adjusted according to the experimental needs. The bottom of the chamber can be optically assessed as well as indicated by the condenser. Depending on the angle of the pipette, the electromagnet (or permanent magnet) is aligned from below without blocking the light pathway. If experiments are to be conducted over extended time periods, the chambers can be covered with silicone sealant to enable gas exchange between compartments and to prevent evaporation of the cell-culture media.
Supplementary information
Supplementary Information
Supplementary Fig. 1
Supplementary Video 1
Nanoparticle mobility in the glass capillary pipette in the presence of a magnetic field.
Supplementary Video 2
Virus stamping of a cultured rat cortical neuron.
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Schubert, R., Herzog, S., Trenholm, S. et al. Magnetically guided virus stamping for the targeted infection of single cells or groups of cells. Nat Protoc 14, 3205–3219 (2019). https://doi.org/10.1038/s41596-019-0221-z
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DOI: https://doi.org/10.1038/s41596-019-0221-z
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