( ...)Effectene Transfection Reagent provides efficient gene delivery ,,, to primary neuronal cell cultures

Jochen Meier, Ivonne Strmel, Radu Iosub, Sonja Schmidt, and Rosemarie Grantyn
Developmental Physiology, Johannes Mller Institute, Humboldt University Medical School (Charit), Berlin, Germany

Efficient delivery of DNA to primary neuronal cell cultures is of critical importance for extending our knowledge of the molecular basis of neuronal physiology. Here, we describe the use of Effectene Transfection Reagent for successful transfection of primary differentiated neurons in rat superior colliculus cultures.

Enhanced green fluorescent protein (EGFP) chimeras have proven to be a powerful tool for studying the underlying cellular mechanisms for dynamic remodeling of synaptic compounds in living neurons (1). However, a major obstacle to the use of such chimeras is an effective gene delivery method. Gene delivery based on non-viral DNA vehicles is an attractive alternative because it can be applied under a wide range of laboratory conditions. We developed a transfection procedure using Effectene Reagent and found it highly suited for easy and efficient transfection of differentiated neurons in primary cultures from rat superior colliculus. Effectene Reagent shows low cytotoxicity and delivers efficient transfection using small amounts of DNA. Furthermore, transfection can be carried out in the presence of serum.

Materials and methods

Superior colliculus neurons were prepared from embryonic day 19 Wistar rats, and cells were plated at a density of 7.5 x 104 cells/cm2 on laminin-coated glass coverslips (16 mm). Cultures were maintained in neurobasal B27 medium supplemented with 1% fetal calf serum. After neuron attachment, coverslips were transferred (cell-side down) to dishes containing a confluent layer of collicular glial cells and were cultured for up to 3 weeks. On the second day in vitro, cytosine arabinoside (4 M) was added to inhibit glial cell proliferation. Transfection was performed on day 9 in vitro using various ratios of EGFP plasmid DNA (carrying a CMV promoter and isolated from DH5alpha E. coli using a QIAGEN Plasmid Kit) to Effectene reagent (Figure 1). Transfection was performed as described in the Effectene Transfection Reagent Handbook. During DNAEnhancer Effectene Reagent complex formation, the coverslips containing the neurons were removed from the glial cell culture and placed cell-side up in 350 l fresh, pre-warmed culture medium in the well of a 24-well plate. Culture medium (350 l per well) was added to transfection complexes and mixed by pipetting up and down. Diluted transfection complexes were then added to neuronal cultures and incubated for 4.5 hours. After incubation, the coverslips were returned to the glial cell culture (cell-side down), and cells were cultured for 24 hours before analysis of expression. All transfections were performed in triplicate. Control cells were exposed to Effectene Reagent complexed with plasmid DNA lacking a CMV eukaryotic promoter. Immunofluorescence was performed as previously described (2).

Efficient Transfection Using Effectene Reagent

Figure 1 Dependence of transfection efficiencies on transfection conditions. Transfection efficiencies are expressed as the percentage of neurons that are GFP-positive. Note that transfection efficiency decreased when more than 5 l of Effectene Reagent was added. Highest transfection efficiencies were obtained using 200 ng of DNA in combination with 25 l of Effectene Reagent per well. For culture and transfection conditions, see Materials and methods.

Briefly, fixed and permeabilized cells were incubated for 1 hour at room temperature with a monoclonal anti-microtubule-associated protein 2 (MAP2) antibody. Cells were washed and incubated with a Cy3-conjugated secondary antibody for 45 minutes at room temperature.

Results and discussion

Figure 1 shows transfection efficiency in randomly chosen view-fields, as determined by counting the number of cell nuclei, MAP2-positive neurons, and GFP-positive fluorescent neurons. The highest transfection efficiency was obtained using 200 ng of DNA with 25 l of Effectene Reagent. Under these conditions, Effectene Reagent is also suitable for transfection of glial cells (data not shown). Use of 10 l Effectene Reagent or exposure of cells to complexes for longer than 5 hours was toxic to neurons. Figure 2 shows two MAP2-positive transfected neurons (green) in a culture that was treated with 200 ng DNA and 5 l of Effectene Reagent. Nuclei (blue) of cells not reacting with MAP2 antibodies are glial-cell nuclei. Note that the nuclei of transfected neurons are not fragmented, which is an indicator of good cell health. In control transfection experiments (i.e., when the plasmid lacking the eukaryotic promoter was used), green fluorescence was not observed.

Transfected Primary Neuronal Cells

Figure 2 Fluorescence micrographs of transfected primary cultures from rat superior colliculus. On day 9 in vitro, cultures were treated with 200 ng of EGFP plasmid DNA complexed with 5 l of Effectene Reagent. Cells were incubated with the complexes for 4.5 hours, the cells were removed, and incubated for a further 24 hours. A Cells were fixed and stained for MAP2-immunoreactivity which specifically labels the somatodendritic compartments of neurons (red). B Cell nuclei were stained using DAPI (blue). C Cells expressing EGFP appear green. D Superimposed images.

Conclusions

• Effectene Transfection Reagent can be used for efficient gene delivery into differentiated neurons in primary cell culture.
  
• Small amounts of DNA together with Effectene Transfection Reagent delivered high transfection efficiencies and expression levels, without damaging neurons.
  
• Cell death in 24-well plates was negligible if less than 10 l Effectene Reagent was used per well and the exposure of cells to transfection complexes was less than 5 hours.
  
• Effectene Transfection Reagent is a powerful tool for in vitro delivery of genes to nondividing cells - particularly differentiated neurons - and therefore greatly facilitates studies of neurophysiology in living cells.

References

1. Meier, J., Vannier, C., Serg, A. Triller, A., and Choquet, D. (2001) Fast and reversible trapping of surface glycine receptors by gephyrin. Nat. Neurosci. 4, 253.

2. Meier, J., Meunier- Durmort, C., Forest, C., Triller, A., and Vannier, C. (2000) Formation of glycine receptor clusters and their accumulation at synapses. J. Cell Sci. 113, 2783.




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