Skip to main content

Advertisement

SpringerLink
Log in

Biolistic transfection of neuronal cultures using a hand-held gene gun

  • Protocol
  • Published:

From Nature Protocols

View current issue Submit your manuscript

Abstract

Biolistic transfection is a technique in which subcellular-sized particles coated with DNA are accelerated to high velocity to propel them into cells. This method is applicable to tissues, cells and organelles, and can be used for both in vitro and in vivo transformations; with the right equipment, it is simple, rapid and efficient. Here we provide a detailed protocol for biolistic transfection of plasmids into cultured human embryonic kidney (HEK) 293 cells and organotypic brain slices using a hand-held gene gun. There are three major steps: (i) coating microcarriers with DNA, (ii) transferring the microcarriers into a cartridge to make a 'bullet', and (iii) firing the DNA-coated microcarriers into cells using a pulse of helium gas. The method can be readily adapted to other cell types and tissues. The protocol can be completed in 1–2 h.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1: Helios gene gun.
Figure 2: Using the gene gun.
Figure 3: HEK293 cells expressing EYFP.
Figure 4: A neuron in a cerebellar brain slice labeled with EYFP.
Figure 5: A neuron in a brain slice expressing two protein subunits.

Similar content being viewed by others

References

  1. Klein, T.M., Wolf, E.D., Wu, R. & Sanford, J.C. High-velocity microprojectiles for delivering nucleic acids into living cells. Nature 327, 70–73 (1987).

    Article  CAS  Google Scholar 

  2. Christou, P., McCabe, D.E. & Swain, W.F. Stable transformation of soybean callus by DNA-coated gold particles. Plant Physiol. 87, 671–674 (1988).

    Article  CAS  Google Scholar 

  3. Lin, M. The gene gun: current applications in cutaneous gene therapy. Int. J. Dermatol. 39, 161–170 (2000).

    Article  CAS  Google Scholar 

  4. Yang, C.H. et al. Seeing the gene therapy: application of gene gun technique to transfect and decolour pigmented rat skin with human agouti signalling protein cDNA. Gene Ther. 11, 1033–1039 (2004).

    Article  CAS  Google Scholar 

  5. O'Brien, J.A. et al. Modification 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 

  6. Jiao, S., Cheng, L., Wolff, J.A. & Yang, N.-S. Particle bombardment mediated gene transfer and expression in rat brain tissues. Biotechnology 11, 497–502 (1993).

    CAS  PubMed  Google Scholar 

  7. Wirth, M.J. & Wahle, P. Biolistic transfection of organotypic cultures of rat visual cortex using a handheld device. J. Neurosci. Methods 125, 45–54 (2003).

    Article  CAS  Google Scholar 

  8. Gerber, H.-P., Dixit, V. & Ferrara, N. Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl2 and A1 in vascular endothelial cells. J. Biol. Chem. 273, 13313–13306 (1998).

    Article  CAS  Google Scholar 

  9. Wellmann, H., Kaltschmidt, B. & Kaltschmidt, C. Optimized protocol for biolistic transfection of brain slices and dissociated cultured neurons with a hand-held gene gun. J. Neurosci. Methods 92, 55–64 (1999).

    Article  CAS  Google Scholar 

  10. Sanford, J.C., Smith, F.D. & Russell, J.A. Optimizing the biolistic process for different biological applications. Methods Enzymol. 217, 483–509 (1993).

    Article  CAS  Google Scholar 

  11. Hargreaves, A.C., Lummis, S.C. & Taylor, C.W. Ca2+ permeability of cloned and native 5-hydroxytryptamine type-3 receptors. Mol. Pharmacol. 46, 1120–1126 (1994).

    CAS  PubMed  Google Scholar 

  12. Stoppini, L., Buchs, P.A. & Muller, D. A simple method for organotypic cultures of nervous tissue. J. Neurosci. Methods 37, 173–182 (1991).

    Article  CAS  Google Scholar 

  13. Shaw, G., Morse, S., Ararat, M. & Graham, F.L. Preferential transformations of human neuronal cells by human adenoviruses and the origin of HEK-293 cells. FASEB J. 16, 869–871 (2002).

    Article  CAS  Google Scholar 

  14. Wirth, M.J., Patz, S. & Wahle, P. Transcellular induction of neuropeptide Y expression by NT4 and BDNF. Proc. Natl. Acad. Sci. USA 102, 3064–3069 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Medical Research Council and the Wellcome Trust. S.C.R.L. holds a Wellcome Trust Senior Research Fellowship in Basic Biomedical Science.

Author information

Authors and Affiliations

  1. Neurobiology Division, Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK

    John A O'Brien & Sarah C R Lummis

  2. Department of Biochemistry, University of Cambridge, Cambridge, CB2 1QW, UK

    Sarah C R Lummis

Authors
  1. John A O'Brien
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sarah C R Lummis
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sarah C R Lummis.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

O'Brien, J., Lummis, S. Biolistic transfection of neuronal cultures using a hand-held gene gun. Nat Protoc 1, 977–981 (2006). https://doi.org/10.1038/nprot.2006.145

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2006.145

  • Springer Nature Limited

This article is cited by

Search

Navigation