Gene Expression in Adherent HEK293 Cells
This is intended as a guide only; for full experimental details please read the references provided.
In Brief Download the PDF of this protocol
Protocol for transient gene expression in adherent HEK293 cells using PEI STAR™ transfection reagent .
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.
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