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High Ca2+-phosphate transfection efficiency in low-density neuronal cultures

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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Figure 1: Flowchart of the main procedures of our Ca2+-phosphate transfection protocol.
Figure 2: Formation and subsequent dissolution of DNA-Ca2+-phosphate precipitate.
Figure 3: High transfection efficiency achieved with our improved protocol in low-density hippocampal cultures.
Figure 4: Transfection of single autaptic neurons in microisland cultures.
Figure 5: Successful transfection of both mature and immature neurons.
Figure 6: Transfection efficiency in glial cells is very low.

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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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Correspondence to Gong Chen.

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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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