Protocol for Transient Gene Expression in Adherent HEK293 Cells using PEI STAR™ Transfection Reagent

This protocol is intended as a guide only, for full experimental details please read the reference provided.

In Brief

PEI STAR™ (Cat. No. 7854) is a chemically defined, high-performance polyethylenimine (PEI) transfection reagent for cost-effective, affordable and scalable transient gene expression. PEI is a synthetic polymer with an exceptionally high positive charge density in pH-neutral solutions. Positively charged PEI binds strongly to negatively charged DNA and imparts a net cationic charge, allowing the DNA to enter cells. PEI is a non-viral vector commonly used to transfect HEK293 and CHO cells. Applications include production of recombinant proteins, antibodies and viruses.

Pre-dissolved, ready-to-use formulation of PEI STAR™ also available: PEI STAR-Go™ transfection reagent (Cat. No. 8174).

Materials

  • PEI STAR™, 1 mg/mL, pH neutralized, sterile-filtered.
  • HEK293 expression medium or DMEM maintained at 37°C. Transfection is inhibited by serum. Only use media that is reduced-serum, serum-free or chemically defined.
  • HEK293 cells, ~50,000 cells/cm2, seeded the day prior to transfection.
  • Transfection-grade plasmid DNA (pDNA) with gene of interest, 1 μg per 106 cells to be transfected.

General Guidance

We list recommended reagent quantities per 106 cells in the table below. If performing many very similar transfections (e.g. making many similar viruses), we would recommend co-varying on these parameters over optimization ranges first.

Parameter Recommended Optimization Range
PEI STAR™ Concentration (per 106 cells) 3.0 μg 2.0 to 4.0 μg
DNA Concentration (per 106 cells)  1.5 μg 1.0 to 2.0 μg
PEI STAR™/DNA Complex Time 10.0 min 5.0 to 15.0 min

Even if performing co-transfection, keep the total DNA concentration range the same (1.0 to 2.0 μg/106 cells).

If high toxicity is observed, then reduce the quantity of PEI and/or DNA first. We do not recommend replacing the media post-transfection to reduce toxicity except as a last resort.

Before Transfection

Seed the cells the day before transfection to reach 50-80% confluence on the day of transfection. Seed approximately 50,000 cells per cm2.

Transfection

  1. Measure the cell density to determine the transfection parameters based on the number of viable cells.
  2. Dilute 1.0 μg pDNA per 106 cells in 5% final culture volume of DMEM or media. Mix well.
  3. Dilute 3.0 μg PEI STAR™ per 106 cells in 5% final culture volume of DMEM or media. Mix well.
  4. Mix the solution of pDNA and PEI STAR™ together by quickly and briefly vortexing or inverting the tube several times.
  5. Allow the mixture of pDNA and PEI STAR™ to rest for 10 minutes at room temperature.
  6. Gradually add the pDNA and PEI STAR™ mixture to the cells dropwise while swirling the plate.
  7. Incubate the cells at typical incubation conditions.

References

Boussif et al (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc.Natl.Acad.Sci.U.S.A. 92 7297. PMID: 7638184.

Longo et al (2013) Transient mammalian cell transfection with polyethylenimine (PEI). Methods Enzymol. 529 227. PMID: 24011049.

Support

Support is available by emailing us at techsupport@bio-techne.com.

Frequently Asked Questions 

  • What are common troubleshooting tips if transfection efficiency is low?

    If transfection efficiency is low, check the quality and purity of the DNA. It is also advisable to adjust the PEI-to-DNA ratio to improve complex formation. Additionally, optimizing cell confluency, with a recommended range of 60 to 80%, can enhance results. Ensuring the proper incubation time for the DNA-PEI complexes before applying them to the cells is another important factor to consider.

  • Is PEI STAR™ cytotoxic to HEK293 cells?

    When used at recommended concentrations, PEI STAR™ exhibits low cytotoxicity. Higher concentrations or improper ratios can increase toxicity, so optimization is important.

  • Can PEI STAR™ transfect other cell types?

    While optimized for HEK293 cells, PEI STAR™ reagent can be tested on other adherent mammalian cells, but transfection efficiency and toxicity should be evaluated per cell line.