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Efficient transfection of DNA or shRNA vectors into neurons using magnetofection

Nature Protocols volume 2, pages 3090–3101 (2007)Cite this article

Abstract

Efficient and long-lasting transfection of primary neurons is an essential tool for addressing many questions in current neuroscience using functional gene analysis. Neurons are sensitive to cytotoxicity and difficult to transfect with most methods. We provide a protocol for transfection of cDNA and RNA interference (short hairpin RNA (shRNA)) vectors, using magnetofection, into rat hippocampal neurons (embryonic day 18/19) cultured for several hours to 21 d in vitro. This protocol even allows double-transfection of DNA into a small subpopulation of hippocampal neurons (GABAergic interneurons), as well as achieving long-lasting expression of DNA and shRNA constructs without interfering with neuronal differentiation. This protocol, which uses inexpensive equipment and reagents, takes 1 h; utilizes mixed hippocampal cultures, a transfection reagent, CombiMag, and a magnetic plate; shows low toxicity and is suited for single-cell analysis. Modifications done by our three laboratories are detailed.

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Figure 1: Expression of EGFP in neuronal cultures (Method C).
Figure 2: Demonstration of the efficiency of magnetofection using an EGFP construct and neurons cultured according to Method A.
Figure 3: Expression of PICK1 and α7 nAChR by magnetofection in cultured hippocampal GABAergic interneurons and pyramidal cells (Method A).
Figure 4: GFP and GFP-gephyrin expression in young and mature neurons (method B).
Figure 5: Knockdown of SynGAP expression using specific RNAi (Method C).

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Acknowledgements

We thank Grigory Krapivinsky (Harvard Medical School, Boston) for help with preparing of shRNA coding vector. We are grateful to Corinne Sidler, Susanne Erb-Vögtli and Dubravka Göckeritz-Dujmovic (University of Zürich) for excellent technical assistance. This study was supported by grants from the Swiss National Science Foundation (to C.F. and J.-M.F.), the Swiss Foundation for Research on Muscle Diseases (to C.F.), INSERM (to C.P. and I.M.) and the French Foundation for Medical Research (to I.C.).

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Author notes

  1. Ilona Chudotvorova & Christian Fuhrer

    Present address: Present address: Department of Psychiatry, Mount Sinai Medical Center, One Gustave Levy Place, New York, New York 10029, USA (I.C.); Open Access Coordination, Research Library Irchel, Main Library, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland (C.F.).,

  2. Thomas Buerli and Christophe Pellegrino: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Neurochemistry, Brain Research Institute, University of Zürich, Winterthurerstrasse 190, Zürich, CH-8057, Switzerland

    Thomas Buerli & Christian Fuhrer

  2. Institute of Pharmacology and Toxicology, University of Zürich, Winterthurerstrasse 190, Zürich, CH-8057, Switzerland

    Thomas Buerli, Barbara Lardi-Studler & Jean-Marc Fritschy

  3. INMED/INSERM Unite 29, 163 Route de Luminy, Marseille, 13009, France

    Christophe Pellegrino, Ilona Chudotvorova & Igor Medina

  4. School of Medicine, Institute of Life Science, Swansea University, Singleton Park, Swansea, SA2 8PP, UK

    Kristin Baer

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Correspondence to Igor Medina.

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Buerli, T., Pellegrino, C., Baer, K. et al. Efficient transfection of DNA or shRNA vectors into neurons using magnetofection. Nat Protoc 2, 3090–3101 (2007). https://doi.org/10.1038/nprot.2007.445

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