Cultured neurons are among the most difficult cells to transfect due to their sensitivity to microenvironmental changes. While calcium phosphate is widely used to transfect neurons due to its low toxicity (Goetze et al. 2004; Holz et al. 2000; Jiang and Chen 2006; Micheva et al. 2003; Passafaro et al. 2003; W. G. Chen et al. 2003; Xia et al. 1996), transfection efficiencies are extremely low compared to those of other methods (~1–5%, on average) (Craig 1998; Washbourne and McAllister 2002; Xia et al. 1996). To address these shortcomings, researchers used our CalPhos Mammalian Transfection Kit to develop a novel calcium phosphate-based transfection method for cultured neurons that increases transfection efficiency tenfold while maintaining low toxicity (Figure 1). These improvements were accomplished by two key modifications: first, the DNA-Ca²⁺ and phosphate solutions are gently mixed to obtain a fine, homogeneous precipitate. Second, the precipitate is dissolved in a slightly acidic cell culture medium to reduce its toxicity. Combined, these modifications provide a high-efficiency, low-toxicity protocol that enables the ready transfection of single autaptic neurons as well as mature neurons (Jiang and Chen 2006).

Figure 1. Flowchart of the Ca²⁺-phosphate transfection protocol.

Formation of a homogeneous DNA-Ca²⁺-phosphate precipitate

As it is believed that the DNA-Ca²⁺-phosphate precipitate enters cells through endocytosis, followed by some DNA molecules making their way to the nucleus, the authors first looked to the precipitate formed. They found that the continuous vortexing used in other protocols often results in large precipitate particles with uneven distribution (Figure 2, Panel A). As these particles are not endocytosed efficiently by neurons, the authors opted for a gradual mixing of the DNA-CaCl₂ and 2X HBS solutions. When coupled with very mild (rather than continuous) vortexing, researchers were able to consistently form small, easily-endocytosed precipitate particles (Figure 2, Panels B and C).

Figure 2. Formation and subsequent dissolution of the DNA-Ca²⁺-phosphate precipitate. Panel A. Continuous vortexing when mixing DNA with Ca²⁺ and phosphate buffer results in large clusters of precipitate (image taken after 1 hr incubation). Panels B and C. Formation of an optimal DNA-Ca²⁺-phosphate precipitate through gentle vortexing during mixing (image taken after 1 hr incubation). Panel D. Dissolution of the precipitate with slightly acidic transfection medium pre-equilibrated in a 10% CO₂ incubator. Scale bar = 50 mm.

Improved dissolution of the DNA-Ca²⁺-phosphate precipitate

A critical parameter for this protocol is the dissolution of the DNA-Ca²⁺-phosphate precipitate following the transfection incubation period. While most protocols recommend simply washing the cells with transfection medium, it was found that this was insufficient to remove the excess precipitate.

To solve this problem, serum-free transfection medium was slightly acidified by pre-equilibrating it in a 10% CO₂ incubator. This acidification helps to dissolve the precipitate, significantly reducing neuronal toxicity and allowing the cells to be incubated with the precipitate for a much longer period of time (Figure 2, Panel D).

Finally, rather than performing transfections in the same plate that neurons were cultured on, transfections were done in a new, separate culture plate. Neurons were plated on coverslips, transferred to a new culture plate for transfection, and then returned to their original culture plate and medium post-transfection.

High-efficiency transfection of low-density neuronal cultures

Due to these improvements, the authors achieved transfection efficiencies of up to 60% in low-density primary neuronal cultures with incubation times of 45 min–3 hr (Figure 3). One microisland contained a total of 22 neurons, of which ~80% (17 neurons) were transfected. The entire coverslip contained 127 transfected neurons out of a total of 211, yielding a transfection rate of ~60.2%. In contrast, previous reports obtained an average transfection efficiency of 1–5% (Köhrmann et al. 1999; Watanabe et al. 1999; Xia et al. 1996). Significantly, exogenous gene expression was both stable and rapid: GFP expression was observed within 4 hours of transfection and persisted for more than 7 days. These features allowed the authors to use this method to study endophilin function in synaptic vesicle endocytosis (Y. Chen et al. 2003).

Figure 3. High transfection efficiency is achieved in low-density hippocampal cultures with our improved protocol. Phase contrast (Panels A–C) or fluorescent (Panels D–F) microscopy was used to capture images of GFP-transfected cells in three independent transfections. Neurons were cultured for 10–15 days according to previously described methods (Chen, Haratas, and Tsien 2004; Jiang, Deng, and Chen 2004). Figures are reproduced from Jiang, Deng, and Chen 2004 and Jiang and Chen 2006.

Transfection of mature neurons in culture

Calcium phosphate methods are often used for transfecting young neurons (2–10 days in culture). Mature neurons are more difficult to transfect due to their tendency to die shortly after transfection. This improved protocol enabled the successful transfection of not only young (Figure 4, Panels A and B) but also mature neurons, allowing for the analysis of cells that have well-established synaptic networks (e.g., cells that have been grown for more than 20 days in culture) (Figure 4, Panels C and D). The new method allowed for the successful transfection of 82-day-old neurons (Figure 4, Panel D), a remarkable achievement given that neurons rarely survive longer than 3 months in culture.

Figure 4. Successful transfection of both immature and mature neurons. Panels A and B. Transfection of young neurons (2 and 7 days in vitro [d.i.v.]) with EGFP. Panels C and D. Transfection of mature neurons (24 and 82 d.i.v.) with EGFP. Scale bar = 50 mm. Figures are reproduced from Jiang and Chen 2006.

High neuronal and low glial transfection efficiency

The authors' neuronal cultures contained many glial cells due to neurons being plated on a monolayer of cortical astrocytes. Excessive astrocyte transfection is undesirable, as it can generate high background signal (e.g., GFP) or could affect neuronal function through neuron-glia interactions. Notably, this method results in a low rate of glial transfection (Figure 5). Transfections were also effective for Banker-type cultures, where neuron-glial contact is minimal. This may be due to the presence of cytosine arabinoside, a reagent that stops glial proliferation and may also suppress transfection. Combined, this protocol provided transfection efficiencies in low-density neuronal cultures of 25.2% (grown with astrocytes) or 21.6% (grown without astrocytes) (Figure 5, Panels A and B). Finally, the authors demonstrated the compatibility of this protocol with several different constructs, including Rab3a and dynamin (Figure 5, Panels C and D).

Figure 5. Transfection efficiency in glial cells is very low. Panel A. Only a few glial cells (arrowhead) are transfected in neuronal cultures with a monolayer of astrocytes. Panel B. In Banker-type cultures where neurons usually do not contact glial cells directly, transfection efficiency remains high (21.6%). Panels C and D. Successful transfections of EGFP-Rab3a (Panel C) and EGFP-dynamin (Panel D) demonstrate the compatibility of this protocol with multiple different constructs. Figures are reproduced from Jiang and Chen 2006.

Combined, these results demonstrate that this protocol, powered by our CalPhos Mammalian Transfection Kit, allows the highly efficient transfection of cultured neurons with low toxicity. The key features of this method are a carefully produced, fine DNA-Ca²⁺-phosphate precipitate and subsequent dissolution of the precipitate with a short exposure to a slightly acidic medium. This protocol is compatible with a variety of plasmid constructs and allows the successful transfection of both mature and immature neurons. Its high efficiency allows even single autaptic neurons to be transfected for subsequent analyses. In addition to our CalPhos Mammalian Transfection Kit, we also offer a wide range of In-Fusion Cloning products that enable you to generate your transfection construct quickly, easily, and without subcloning steps.

Full-length methods and materials are available from the author's original manuscript (Jiang and Chen 2006).

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