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In vivo single-cell electroporation for transfer of DNA and macromolecules

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Abstract

Single-cell electroporation allows transfection of plasmid DNA or macrocmolecules into individual living cells using modified patch electrodes and common electrophysiological equipment. This protocol is optimized for rapid in vivo electroporation of Xenopus laevis tadpole brains with DNA, dextrans, morpholinos and combinations thereof. Experienced users can electroporate roughly 40 tadpoles per hour. The technique can be adapted for use with other charged transfer materials and in other systems and tissues where cells can be targeted with a micropipette. Under visual guidance, an electrode filled with transfer material is placed in a cell body–rich area of the tadpole brain and a train of voltage pulses applied, which electroporates a nearby cell. We show examples of successfully electroporated single cells, instances of common problems and troubleshooting suggestions. Single-cell electroporation is an affordable method to fluorescently label and genetically manipulate individual cells. This powerful technique enables observation of single cells in an otherwise normal environment.

*Note: In the version of this article initially published online, the article’s page numbers should have been 1267–1272. This error has been corrected in the PDF version of the article.

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Figure 1: Illustration of SCE in the optic tectum of the Xenopus laevis central nervous system.
Figure 2: The single-cell electroporation setup.
Figure 3: Electrode loading syringe.
Figure 4: Anticipated results of single-cell electroporation.
Figure 5: Examples of electroporation results requiring troubleshooting.

Change history

  • 30 November 2006

    In the version of this article initially published online, the article’s page numbers should have been 1267–1272. This error has been corrected in the PDF version of the article.

References

  1. Fraser, S.E. Iontophoretic dye labeling of embryonic cells. Methods Cell Biol. 51, 147–160 (1996).

    Article  CAS  Google Scholar 

  2. Driscoll, M. Methods for the study of cell death in the nematode Caenorhabditis elegans . Methods Cell Biol. 46, 323–353 (1995).

    Article  CAS  Google Scholar 

  3. O'Brien, J.A., Holt, M., Whiteside, G., Lummis, S.C. & Hastings, M.H. Modifications to the hand-held Gene Gun: improvements for in vitro biolistic transfection of organotypic neuronal tissue. J. Neurosci. Methods 112, 57–64 (2001).

    Article  CAS  Google Scholar 

  4. Ehrengruber, M.U. et al. Gene transfer into neurons from hippocampal slices: comparison of recombinant Semliki Forest Virus, adenovirus, adeno-associated virus, lentivirus, and measles virus. Mol. Cell Neurosci. 17, 855–871 (2001).

    Article  CAS  Google Scholar 

  5. Liu, Y., Fong, S. & Debs, R.J. Cationic liposome-mediated gene delivery in vivo . Methods Enzymol. 373, 536–550 (2003).

    Article  CAS  Google Scholar 

  6. Lee, T. & Luo, L. Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22, 451–461 (1999).

    Article  CAS  Google Scholar 

  7. Feng, G. et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41–51 (2000).

    Article  CAS  Google Scholar 

  8. Colosimo, A. et al. Transfer and expression of foreign genes in mammalian cells. Biotechniques 29, 314–324 (2000).

    Article  CAS  Google Scholar 

  9. Washbourne, P. & McAllister, A.K. Techniques for gene transfer into neurons. Curr. Opin. Neurobiol. 12, 566–573 (2002).

    Article  CAS  Google Scholar 

  10. Ho, S.Y. & Mittal, G.S. Electroporation of cell membranes: a review. Crit. Rev. Biotechnol. 16, 349–362 (1996).

    Article  CAS  Google Scholar 

  11. Tsong, T.Y. Electroporation of cell membranes. Biophys. J. 60, 297–306 (1991).

    Article  CAS  Google Scholar 

  12. Golzio, M., Teissie, J. & Rols, M.P. Direct visualization at the single-cell level of electrically mediated gene delivery. Proc. Natl. Acad. Sci. USA 99, 1292–1297 (2002).

    Article  CAS  Google Scholar 

  13. Haas, K., Sin, W.C., Javaherian, A., Li, Z. & Cline, H.T. Single-cell electroporation for gene transfer in vivo . Neuron 29, 583–591 (2001).

    Article  CAS  Google Scholar 

  14. Sin, W.C., Haas, K., Ruthazer, E.S. & Cline, H.T. Dendrite growth increased by visual activity requires NMDA receptor and Rho GTPases. Nature 419, 475–480 (2002).

    Article  CAS  Google Scholar 

  15. Sakmann, B. & Neher, E. Patch clamp techniques for studying ionic channels in excitable membranes. Annu. Rev. Physiol. 46, 455–472 (1984).

    Article  CAS  Google Scholar 

  16. Rae, J.L. & Levis, R.A. Single-cell electroporation. Pflugers Arch. 443, 664–670 (2002).

    Article  CAS  Google Scholar 

  17. Rathenberg, J., Nevian, T. & Witzemann, V. High-efficiency transfection of individual neurons using modified electrophysiology techniques. J. Neurosci. Methods 126, 91–98 (2003).

    Article  Google Scholar 

  18. Hashimoto-Torii, K. et al. Differential activities of Sonic hedgehog mediated by Gli transcription factors define distinct neuronal subtypes in the dorsal thalamus. Mech. Dev. 120, 1097–1111 (2003).

    Article  CAS  Google Scholar 

  19. Lovell, P., Jezzini, S.H. & Moroz, L.L. Electroporation of neurons and growth cones in Aplysia californica . J. Neurosci. Methods 151, 114–120 (2006).

    Article  Google Scholar 

  20. Umeda, T., Ebihara, T. & Okabe, S. Simultaneous observation of stably associated presynaptic varicosities and postsynaptic spines: morphological alterations of CA3-CA1 synapses in hippocampal slice cultures. Mol. Cell. Neurosci. 28, 264–274 (2005).

    Article  CAS  Google Scholar 

  21. Yang, Z.J. et al. Novel strategy to study gene expression and function in developing cerebellar granule cells. J. Neurosci. Methods 132, 149–160 (2004).

    Article  CAS  Google Scholar 

  22. Bhatt, D.H., Otto, S.J., Depoister, B. & Fetcho, J.R. Cyclic AMP-induced repair of zebrafish spinal circuits. Science 305, 254–258 (2004).

    Article  CAS  Google Scholar 

  23. Nieuwkoop, P.D. & Faber, J. Normal Table of Xenopus laevis (Daudin) (Elsevier-North Holland Publishing Company, Amsterdam, 1956).

    Google Scholar 

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Correspondence to Hollis T Cline.

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Bestman, J., Ewald, R., Chiu, SL. et al. In vivo single-cell electroporation for transfer of DNA and macromolecules. Nat Protoc 1, 1267–1272 (2006). https://doi.org/10.1038/nprot.2006.186

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