<b>Transfection Of Primary Neuronal Cells</b><br/><br/>

Transfection Of Primary Neuronal Cells

by Xiaodong Wang, Jiang Ning

Introduction

Figure 1. Effects of cytotoxicity on protein expression. E18 Cortex cells were seeded 20,000 cells/well on a poly-D-lysine coated 96-well plate. 0.25 mg gWiz–LacZ/well were transfected with various amount of NeuroFECT into the cells. The Cyototoxicity (blue line) and b-galactosidase (red bar) assay were performed 24-hours post-transfection.
Click here to enlarge.

Primary neuronal cells are known to be difficult to transfect. Sensitivities to media conditions and cytotoxicity of transfection reagents both contribute to the low transfection efficiency, low cell viability, and neurodegeneration that have plagued primary neuronal transfection procedures for many years.

NeuroFECT, a polymer from Genlantis (San Diego, CA), is a transfection reagent specifically designed for transfection efficiency in primary neuronal cells. Optimal transfection protocols are established for each type of primary neuronal cell. In some cells, transfection efficiency is maximized to 40-60%.

NeuroFECT reagent is based on a proprietary cationic polymer. During transfection, the polymer and DNA complexes (polyplexes) are endocytosed into the cells, where the polymer is biodegraded into small non-toxic molecules. (Other cationic polymers tend to accumulate in the cell nucleus, which results in their being toxic.⁽¹⁾) The biodegradability of NeuroFECT dramatically reduces its cytotoxicity and therefore maximizes the delivery of macromolecules into cells.

Materials and methods

Cell preparations. E18 rat cortical and hippocampal cells (Genlantis) were plated at various densities in poly-D-lysine pre-coated 96 and 24-well tissue culture plates using plating medium (Neurobasal/B27/0.5 mM glutamine/25 μM glutamate media). The cells were cultured at 37 C in 5% CO₂ for 3 days. Half the volume of the plating media per well was then removed and replaced with same amount of culture medium (Neurobasal/B27/0.5 mM glutamine).^(2,3)

Figure 2. Effects of different cell numbers on transfection efficiency. E18 cortex cells 15,000-35,000/well were plated on a poly-D-lysine coated 96-well tissue culture plate. 0.25 mg gWiz–LacZ/well was transfected with various amounts of NeuroFECT into the cells. b-galactosidase assays were performed 24-hours post-transfection. Click here to enlarge.

Figure 3. Effects of different ratios of NeuroFECT (mg):DNA (mg) on transfection
efficiency. 20,000 cells/well of E18 cortex cells were plated at poly-D-lysine coated
96-well plate. 0.15 mg – 0.5 mg of gWiz–LacZ/well were transfected with various amounts of NeuroFECT into the cells. The ratio of NeuroFECT (mg):DNA (mg) was 10:1-3:1. The b-galactosidase assay was performed 24-hours post-transfection. Click here to enlarge.

Transfections. Transfection was performed on day 6 or 7 by using various ratios of NeuroFECT (μg)/DNA (μg). Briefly, diluted NeuroFECT and DNA were prepared in separated tubes. Diluted NeuroFECT was added to the diluted DNA in a drop-wise fashion and was mixed with gentle pipetting. The NeuroFECT/DNA complex was incubated for 15-20 minutes at room temperature to form polyplexes. Old culture medium from cells was removed and replaced with fresh culture medium. The NeuroFECT/DNA complex was added to the cells and gently mixed by swirling plate. The cells were cultured at 37 C in 5% CO₂ for 24-48 hours before being assayed.

Cytotoxicity assay. Adenylate kinase (AK) level was measured using the Toxilight Non-destructive Cytotoxicity Bioassay kit (Cambrex Corp., Rockland, ME). AK is released into culture medium when cells die. AK phosphorylates ADP to form ATP. The level of ATP was measured using the bioluminescent firefly luciferase reaction. Briefly, 40 μl of AK detection reagent from the kit was added into 5 μl of culture supernatant medium from transfected cells. The reaction was incubated at room temperature for 5 minutes. The AK level was measured using a luminometer.

β-galactosidase assay.β -galactosidase expression level was measured with a β-galactosidase Assay Kit (Genlantis). The culture medium from gWiz-LacZ (Genlantis) transfected cells was removed. The cells were lysed with 50 μl ࡧ lysis buffer from the kit with quick freeze and thaw. Serial dilutions of β-galactosidase (E. coli) standards were prepared in 50 μl/well. 100 μl of ONPG substrate solution was added into the cell lysates and standards. The samples were incubated at room temperature for 5 - 30 minutes. The absorbance of β-galactosidase was read at 405 nm with a spectrophotometer.

Transfection efficiency assay. The primary neuronal cells were transfected with gWiz-GFP (Genlantis) for 48 hours. The cells were washed with ࡧ PBS and treated with Detachin (Genlantis) to suspend the cells. The percentage of GFP-positive cells was measured with FACS.

Immunofluoresence. The cells were seeded on the poly-D-lysine coated cover slips. gWiz-GFP was transfected into the cells with NeuroFECT reagent for 48 hours. The cells were fixed with 3.7% formaldehyde at room temperature for 20 minutes. The coverslip was rinsed with PBS and mounted with mounting buffer. GFP-positive cells were observed and photographed under fluorescent microscopy.

Results and discussion

Cytotoxicity of NeuroFECT was measured as Adenylate Kinase (AK) level. When cytolysis increases, the amount of AK in the supernatant also increases, which results in emission of higher light intensity. Neuronal cells were plated and transfected as described in the materials and methods. The culture medium was used for AK level assay and the transfected cells were used for β-galactosidase assay. A higher level of light intensity (RLU) indicated that more AK released into the culture medium, and that more cell death happened during the transfection. NeuroFECT showed lower cytotoxicity in most of the transfection conditions (Figure 1). The β-galactosidase expression levels suggest that the cells with higher protein expression had lower cytotoxicity.

Figure 4. The transfection efficiency of NeuroFECT in primary neuronal cells. E18 rat cortical cells (85,000 cells/well) and E18 rat hippocampal cells (65,000 cells/well) were plated on poly-D-lysine coated 24-well tissue culture plates and cover slips for 5 days. 1.0 mg of gWiz–GFP was transfected with various amounts of NeuroFECT into E18 rat cortical and hippocampal cells. The GFP positive cells were measured by FACS (A). Immunofluorescent photos (B) were taken 48-hours post-transfection.
Click here to enlarge.

Optimization of expression levels

Determine the cell numbers for the transfection. Suitable cell numbers can maximize the transfection efficiency. E18 cortex cells were seeded at densities 15,000/well - 35,000/well in a poly-D-lysine coated 96-well plate. The neuronal cells were transfected as described in the materials and methods. The highest protein expression was obtained at 20,000/well of cell density (Figure 2).

Determine the best ratio of NeuroFECT (μg):DNA (μg) for the transfection. Plasmid gWiz-LacZ was transfected into the cells with various amounts of NeuroFECT in the presence of B27 supplement. The NeuroFECT (μg):DNA (μg) ratio ranged from 10:1 to 3:1. β-galactosidase expression levels were measured 24-hours post-transfection. The highest protein expression was obtained at 7:1 NeuroFECT (μg):DNA (μg) in presence of B27 supplement (Figure 3). The data indicated that the ratio of NeuroFECT (μg):DNA (μg) was critical for transfection efficiency. For the initial experiment, it is important to test the ratios from 8:1 to 4:1.

Achieving 40-60% transfection efficiency in E18 rat cortical and hippocampal cells

Plasmid gWiz-LacZ was transfected into E18 rat hippocampal and E18 cortical cells with NeuroFECT. FACS data showed the highest transfection efficiency using NeuroFECT in primary neurons was 64% (Figure 4A). Primary neuronal cells are very delicate. Careful handing of the cells was important for maximizing transfection efficiency. The GFP-positive cells in Figure 4B clearly showed both cell bodies and axons. The cells have good morphology and are networking with each other.

About the authors

Xiaodong Wang and Jiang Ning are scientists at Genlantis, a Division of Gene Therapy Systems, Inc. in San Diego, California.

More information about NeuroFECT and transfection of neuronal cells is available from:
Genlantis
888-428-0558
www.genlantis.com

References

1. Godbey, W.T., Wu, K.K. and Mikos, A.G. Poly (ethylenimine) and its role in gene delivery. J Control Release 60(2-3):149-60 (1999).

2. Brewer, G.J., Torricelli, J.R., Evege, E.K. and Price, P.J. Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J Neurosci Res 35(5):567-76 (1993).

3. Brewer, G.J. and Price, P.J. Viable cultured neurons in ambient carbon dioxide and hibernation storage for a month. Neuroreport 7(9):1509-1512 (1996).