Polyethylenimine (PEI) transfection is a method used to introduce genetic material into eukaryotic cells. PEI is a cationic polymer, and its positive charge allows it to bind with negatively charged nucleic acids, such as plasmid DNA. This binding forms small, condensed particles called PEI-DNA complexes, which are taken up by cells through endocytosis.
Inside the cell, PEI helps the DNA escape cellular compartments and reach the nucleus, where its genetic instructions are read. This technique is widely used because it is effective and more affordable than many alternative gene delivery methods. Its versatility makes it suitable for various applications, from small-scale protein expression to virus production for gene therapy research.
Reagent and Cell Preparation
Proper preparation of the PEI stock solution is foundational for successful transfection. This involves dissolving PEI powder in water and adjusting the pH to approximately 7.0 to maintain cell health. The solution is then sterilized through a 0.22 µm filter to remove microbial contaminants. To prevent degradation, the sterile solution is divided into single-use volumes and stored at -20°C.
The quality of the plasmid DNA is another determinant of transfection success. Use high-purity, endotoxin-free plasmid DNA, as endotoxins are toxic to eukaryotic cells and reduce efficiency. The DNA concentration should be accurately measured, with working concentrations falling within a range suitable for complex formation.
Cell health and density at transfection directly influence the outcome. Plate adherent cells the day before the experiment so they are 50-70% confluent on the day of transfection. This confluency ensures cells are in an active growth phase and can efficiently take up the PEI-DNA complexes. Over-confluent, unhealthy, or high-passage cells will likely yield poor results.
Step-by-Step Transfection Protocol
Begin by separately diluting the plasmid DNA and the PEI stock solution. In one tube, dilute the DNA in a serum-free medium, such as Opti-MEM; serum components can interfere with complex formation. In a separate tube, dilute the PEI stock solution in the same serum-free medium.
Add the diluted PEI to the diluted DNA. This mixture allows for the self-assembly of PEI-DNA complexes. Incubate the mixture at room temperature for 15 to 30 minutes to ensure the complexes are fully formed.
Following incubation, add the complex mixture drop-wise to the cells. This distributed addition prevents localized toxicity. Return the plate to the incubator, allowing the cells to take up the complexes.
To minimize cytotoxicity, replace the medium 4 to 6 hours later or the following day. Wash the cells with phosphate-buffered saline (PBS) before adding fresh, complete growth medium with serum. This supports cell recovery and allows gene expression over the next 24 to 72 hours.
Key Parameters for Optimization
Optimizing several parameters for each cell line and plasmid combination is necessary for success. The primary factors to adjust include:
- PEI to DNA ratio: This is often expressed as a mass ratio (µg of PEI to µg of DNA). An improper ratio can lead to low efficiency or high cell toxicity. Test a range of ratios to find the balance that yields the highest gene expression with the lowest cell death.
- Total plasmid DNA: While more DNA might seem to increase expression, excessive amounts can trigger cellular stress and toxicity. It is necessary to test different DNA quantities to find the optimal concentration that maximizes protein production without harming the cells.
- Type of PEI: Linear PEI is considered less toxic but may be less effective in some cell lines than branched PEI. Branched PEI has a higher charge density, which can enhance DNA condensation and interaction with the cell membrane. Experimenting with both may be required.
Post-Transfection Assessment
Evaluate transfection success 24 to 72 hours after the procedure. A common method is using a reporter gene, such as Green Fluorescent Protein (GFP). When the GFP plasmid is expressed, cells will glow green under a fluorescence microscope, and the percentage of fluorescent cells provides a direct measure of efficiency.
Other quantitative methods can also assess gene expression. Luciferase assays offer a highly sensitive measurement of protein activity. Alternatively, techniques like Western blotting or ELISA can detect and quantify the specific protein encoded by the transfected DNA.
When troubleshooting issues like high cell death or low efficiency, revisit the optimization parameters. Significant cell death may suggest the PEI concentration or the PEI:DNA ratio was too high. Low efficiency might be improved by ensuring optimal cell confluency and systematically testing various PEI:DNA ratios.