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Principles and Operational Procedures for PEI Transfection
Polyethylenimine (PEI) is currently the most widely used gene delivery vector and transfection reagent in industrial-scale, large‑volume transient expression of recombinant proteins or viral vectors. PEI is a highly cationic polymer that readily binds to negatively charged DNA molecules, forming stable complexes. In cell lines such as HEK293 and CHO, PEI‑mediated transfection delivers high transfection efficiencies, making it particularly well suited for large‑scale transient expression experiments. Compared with calcium phosphate–based methods, PEI is simpler to prepare, compatible with a broader range of cell types, and offers superior cost‑effectiveness. Moreover, unlike lipid‑based transfection reagents, PEI exhibits lower cytotoxicity. For cell types that are difficult to transfect via conventional transient approaches, PEI also facilitates efficient lentiviral packaging, meeting the needs of stable transfection in most cell lines.
Principle of PEI Transfection
PEI can encapsulate DNA into positively charged nanoparticles, which can bind to negatively charged residues on the cell surface and enter the cell via endocytosis. Once inside, protonation of the amines leads to a massive influx of counterions and a reduction in osmotic potential, facilitating intracellular release of DNA under low-pH conditions. The prevailing view is that unprotonated amines on PEI can uptake protons within membrane‑bound organelles, such as lysosomes, thereby driving further proton influx and triggering osmotic swelling. Meanwhile, electrostatic repulsion between protonated amines induces conformational changes in PEI; the resulting osmotic expansion causes vesicles to release the polymer–DNA complexes into the cytoplasm. Following disassembly of these complexes, DNA is free to translocate into the nucleus.
PEI Transfection Protocol
PEI transfection reagents are easy to use and exhibit excellent compatibility with serum and antibiotics. A typical protocol is outlined below (using HEK293 cells as an example).
① Before transfection, ensure that cell viability exceeds 95% and that the live-cell density is between (1.5–2.0) × 10^6 cells/mL.
② Dilute the live cell density to 1.05 × 10^6 cells/mL;
③ Invert the pDNA and 1 mg/mL Bestop™ PEI linear transfection reagent several times to mix thoroughly;
④ Add 1 μg of pDNA per milliliter of the culture to be transfected into a clean vial. Dilute with fresh medium to a final concentration of 20 μg/mL (5% of the final culture volume).
⑤ For each milliliter of the culture to be transfected, transfer 3 μL of 1 mg/mL Bestop™ PEI linear transfection reagent into a clean vial. Dilute with culture medium to a final concentration of 60 μg/mL (5% of the final culture volume).
⑥ Mix the diluted pDNA with the Bestop™ PEI linear transfection reagent. Invert several times and let stand at room temperature for 10 minutes. Before use, gently invert the sealed container once.
⑦ Transfect 10 mL of the pDNA/PEI mixture into every 90 mL of culture medium. While mixing, gradually add the mixture to the culture medium and incubate the cells.
⑧ After transfection: If enhancers or supplements are used, they can be added at any time up to 6 hours post-transfection. Subculturing can also be performed after 6 hours.
If a GFP control is used, a transfection efficiency exceeding 70% should be observed after 48 hours. For optimized protocols, a transfection efficiency of over 80% is considered reasonable.
Frequently Asked Questions about PEI
Q1: Will preparing and cryopreserving a larger volume of stock solution extend its shelf life?
A: Cryopreservation is not recommended; the storage solution can be kept at 2–8°C for up to 3 months without issue.
Q2: Is it necessary to change the culture medium after transfection?
A: Medium change is not required; you can also assess the cells’ condition 6–8 hours post-transfection to determine whether a medium change is necessary.
Q3: Is serum-free culture medium required during transfection?
A: When preparing the complex, a serum-free culture medium must be used; however, serum-free conditions are not required when adding it to the cells.
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