Abstract
This protocol describes a high-efficiency Ca2+-phosphate transfection method with low cell toxicity. The Ca2+-phosphate transfection method is widely used in transfecting neurons because of its low cell toxicity and simplicity in use, but the efficiency is typically low (∼1–5%). To solve this problem we have developed a new Ca2+-phosphate transfection protocol that increases the efficiency by 10-fold (≤60%), while maintaining low cell toxicity. First, it is critical to have gentle mixing of the DNA-Ca2+ solution with phosphate buffer to form a homogeneous snowlike precipitate (particle size 1–3 μm). Second, the precipitate should be dissolved using a slightly acidic culture medium to reduce cell toxicity. The high efficiency of this new protocol makes it possible to transfect single autaptic neurons as well as mature neurons (15–82 days in vitro) for gene functional analysis. The total time required for the protocol is 2–4 h (including 45 min–3 h incubation time).
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References
Xia, Z., Dudek, H., Miranti, C.K. & Greenberg, M.E. Calcium influx via the NMDA receptor induces immediate early gene transcription by a MAP kinase/ERK-dependent mechanism. J. Neurosci. 16, 5425–5436 (1996).
Micheva, K.D., Buchanan, J., Holz, R.W. & Smith, S.J. Retrograde regulation of synaptic vesicle endocytosis and recycling. Nat. Neurosci. 6, 925–932 (2003).
Chen, W.G. et al. Upstream stimulatory factors are mediators of Ca2+-responsive transcription in neurons. J. Neurosci. 23, 2572–2581 (2003).
Holz, R.W. et al. A pleckstrin homology domain specific for phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P2) and fused to green fluorescent protein identifies plasma membrane PtdIns-4,5-P2 as being important in exocytosis. J. Biol. Chem. 275, 17878–17885 (2000).
Passafaro, M., Nakagawa, T., Sala, C. & Sheng, M. Induction of dendritic spines by an extracellular domain of AMPA receptor subunit GluR2. Nature 424, 677–681 (2003).
Goetze, B., Grunewald, B., Baldassa, S. & Kiebler, M. Chemically controlled formation of a DNA/calcium phosphate coprecipitate: application for transfection of mature hippocampal neurons. J. Neurobiol. 60, 517–525 (2004).
Craig, A.M. Transfecting Cultured Neurons (MIT Press, Cambridge, MA, 1998).
Washbourne, P. & McAllister, A.K. Techniques for gene transfer into neurons. Curr. Opin. Neurobiol. 12, 566–573 (2002).
Chen, Y. et al. Formation of an endophilin-Ca2+ channel complex is critical for clathrin-mediated synaptic vesicle endocytosis. Cell 115, 37–48 (2003).
Chen, G., Harata, N. & Tsien, R.W. Paired-pulse depression of unitary quantal amplitude at single hippocampal synapses. Proc. Natl. Acad. Sci. USA 101, 1063–1068 (2004).
Jiang, M., Deng, L.B. & Chen, G. High Ca2+ phosphate transfection efficiency enables single neuron gene analysis. Gene Ther. 11, 1303–1311 (2004).
Kohrmann, M. et al. Fast, convenient, and effective method to transiently transfect primary hippocampal neurons. J. Neurosci. Res. 58, 831–835 (1999).
Watanabe, S.Y. et al. Calcium phosphate-mediated transfection of primary cultured brain neurons using GFP expression as a marker: application for single neuron electrophysiology. Neurosci. Res. 33, 71–78 (1999).
Bekkers, J.M. & Stevens, C.F. Excitatory and inhibitory autaptic currents in isolated hippocampal neurons maintained in cell-culture. Proc. Natl. Acad. Sci. USA 88, 7834–7838 (1991).
Chen, G. & van den Pol, A.N. Multiple NPY receptors coexist in pre- and postsynaptic sites: inhibition of GABA release in isolated self-innervating SCN neurons. J. Neurosci. 16, 7711–7724 (1996).
Deng, L.B. & Chen, G. Cyclothiazide potently inhibits γ-aminobutyric acid type A receptors in addition to enhancing glutamate responses. Proc. Natl. Acad. Sci. USA 100, 13025–13029 (2003).
Acknowledgements
We thank Rubing Chen for assistance in creating Figure 1. A portion of this material appeared in Gene Therapy 11, 1303–1311 (2004). This work is supported by a grant from the National Science Foundation (0236429) to G.C.
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Jiang, M., Chen, G. High Ca2+-phosphate transfection efficiency in low-density neuronal cultures. Nat Protoc 1, 695–700 (2006). https://doi.org/10.1038/nprot.2006.86
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DOI: https://doi.org/10.1038/nprot.2006.86
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