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Novel Twist on Primary Cell Transfection

Biodegradable Polymer-Based DNA Carrier Designed to Facilitate Process in Mammalian Cells

• Joshua Bryson, Ph.D.

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Results

Figure 1. (A) Luciferase expression (relative light units per mg of protein) assayed 48 hours after transfection; (B) Cell viability—blue bars represent normalized MTT absorbance data. Red bars represent normalized BCA absorbance data. Click Image To Enlarge +

Figure 1. (A) Luciferase expression (relative light units per mg of protein) assayed 48 hours after transfection; (B) Cell viability—blue bars represent normalized MTT absorbance data. Red bars represent normalized BCA absorbance data.

Luciferase Expression and Toxicity Profiles. In HDFn cells, only lipid reagent A and Glycofect elicited a positive response for luciferase expression (Figure 1A) compared with controls. Both lipid B and DNA only showed no transfection. Although some toxic response was observed for each transfection reagent used, minimal toxicity, confirmed by both MTT and BCA assays (Figure 1B), was observed in the Glycofect wells after 48 hours.

Figure 2. Flow cytometry histograms show total enhanced green fluorescent protein expression in HDFn cells after 48 hours; positive EGFP response gate was set based on a cells-only control sample. The X-axis represents event intensity, and the Y-axis represents total events counted. Click Image To Enlarge +

Figure 2. Flow cytometry histograms show total enhanced green fluorescent protein expression in HDFn cells after 48 hours; positive EGFP response gate was set based on a cells-only control sample. The X-axis represents event intensity, and the Y-axis represents total events counted.

EGFP Expression in HDFn. Total gene expression per cell was carried out via flow cytometry using EGFP. Flow cytometry indicated that both lipid reagent A and Glycofect enabled the expression of EGFP beyond the positive threshold gate in over 50% of the cell population. The sample transfected with naked DNA showed no transfection, and lipid reagent B showed low expression (13.3%). Although a higher percentage of cells are expressing with lipid reagent A (58.4%), cells show greater signal intensity in the Glycofect sample (Figure 2). Micrographs of these trials further verify this trend (Figure 3).

Conclusions

Figure 3. Fluorescent micrographs show total EGFP expression in HDFn cells after 48 hours; the differential interference contrast images (top row) and the fluorescent images (bottom row) are shown. Click Image To Enlarge +

Figure 3. Fluorescent micrographs show total EGFP expression in HDFn cells after 48 hours; the differential interference contrast images (top row) and the fluorescent images (bottom row) are shown.

The Glycofect reagent has demonstrated improved DNA transfection in primary cells by showing high gene expression in a high percentage of cells, while maintaining low toxicity.

Reporter gene data shows that Glycofect is an effective transfection reagent in human dermal fibroblast cells and has the potential to be used in therapies requiring transient genetic alteration of this cell type.