Polyethylene imine (PEI) is currently the most widely used gene vector and transfection reagent in the field of industrial and large-scale transient transfection expression of recombinant proteins or viral vectors. PEI is a polymer with highly charged cations that easily binds to negatively charged DNA molecules to form complexes. In HEK293 and CHO cells, PEI transfection method can achieve high transfection efficiency, especially suitable for large-scale transient transfection expression experiments. Compared to the calcium phosphate method, PEI transfection reagents are easier to prepare, suitable for a wider variety of cells, and have higher cost-effectiveness. Compared with transfection reagents such as liposomes, PEI transfection has less toxicity to cells. For cells that are difficult to perform transient transfection, using PEI for lentiviral packaging is also very convenient, meeting the needs of stable transfection for most cells.
PEI transfection principle
PEI can encapsulate DNA into positively charged particles, which can adhere to negatively charged cell surface residues and enter cells through phagocytosis. Once inside the cell, the protonation of amines leads to a large influx of counterions and a decrease in osmotic potential, achieving intracellular release of DNA in a low pH environment. The most widely accepted view is that unpotonated amines on PEI can absorb hydrogen ions in membrane-bound organelles such as lysosomes, which will lead to the influx of more hydrogen ions and induce osmotic swelling. The repulsion between protonated amines causes a conformational change in PEI, and the resulting osmotic expansion causes vesicles to release a complex formed by polymer and DNA into the cytoplasm. After disassembling the complex, DNA can freely fuse into the nucleus.
PEI transfection operation method
PEI transfection reagent is relatively convenient to use and has strong compatibility with serum and antibiotics. The specific operation steps are as follows (taking HEK293 cells as an example)
① Before transfection, ensure that the cell survival rate is greater than 95% and the live cell density is between (1.5-2.0) × 10 ^ 6 cells/mL;
② Dilute the live cell density to 1.05 × 106 cells/mL;
③ Mix pDNA with 1 mg/mL Bestop ™ Invert the PEI linear transfection reagent several times and mix well;
④ Transfer 1 μ g pDNA per milliliter of culture to be transfected into a clean vial. Dilute with fresh culture medium to 20 μ g/mL (5% final culture volume).
⑤ Transfer 3 μ L of 1 mg/mL Bestop per milliliter of culture to be transfected ™ Transfer PEI linear transfection reagent into clean small bottles. Dilute with culture medium to 60 μ g/mL (5% final culture volume).
⑥ Dilute pDNA and Bestop ™ PEI linear transfection reagent mixture. Invert several times and let it stand at room temperature for 10 minutes. Gently invert the sealed container before use.
⑦ Transfect 10 mL of pDNA/PEI mixture every 90 mL of culture medium. Gradually add the mixture to the culture medium while mixing, and incubate the cells.
⑧ After transfection: If enhancers or supplements are used, they can be added at any time after 6 hours of transfection. Passage culture can also be carried out after 6 hours.
If GFP control is used, a transfection rate of over 70% should be observed after 48 hours. For optimized methods, a transfection rate of over 80% is reasonable.
Common problems with PEI usage
Q1: Will configuring more mother liquor for freezing extend its shelf life?
A: It is not recommended to freeze, and storing the solution at 2-8 ℃ for 3 months is not a problem.
Q2: Do we need to change the medium after transfection?
A: It is not necessary to change the medium, and the cell status can be observed 6-8 hours after transfection to determine whether it is necessary to change the medium.
Q3: Is serum-free culture medium necessary for transfection?
A: When preparing the complex, serum-free culture medium is required, while serum-free culture is not required when adding it to cells.
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