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This is intended as a guide only; for full experimental details please read the references provided.
Transient Gene Expression in HEK293 Suspensions using PEI STAR™ Transfection Reagent
Transfection is inhibited by serum. Use media that is reduced-serum, serum-free ("SFM") or chemically deﬁned ("CDM"). Here are some popular media:
|Thermo Fisher Scientific||
Gibco™ Opti-MEM™ I Reduced Serum Media
Gibco™ FreeStyle™ 293 Expression Medium
Gibco™ Expi293™ Expression Medium (with Expi293F™ Cells)
HyClone HyCell TransFX-H
HyClone SFM4Transfx-293 media
|FUJIFILM Irvine Scientific||BalanCD HEK293 (Recommended)|
Cell densities used are for typical HEK293 cultures, with maximum viable cell densities of ~4.0 x 106 cells/mL. If using a high-density system, increase the values for cell density linearly. For example, Expi293™ media and Expi293F™ cells can support viable cell densities over 12 x 106 cells/mL instead of ~4.0 x 106 cells/mL, and so should be transfected at 3.15 x 106 cells/mL instead of 1.05 x 106 cells/mL.
The typical pDNA and PEI concentrations (1.0 and 3.0 mg/L, respectively) can achieve high transfection efﬁciency at viable cell densities from 1.0 x 106 cells/mL up to 5.0 x 106 cells/mL.
If performing the same expression many times, or many similar expressions, we would recommend co-varying on these parameters over corresponding ranges to ﬁnd the optimal conditions:
|PEI Concentration||1.50 to 4.50 mg/(L ﬁnal culture)|
|DNA Concentration||0.75 to 1.50 mg/(L ﬁnal culture)|
|PEI/DNA Complex Time||5.0 to 15.0 minutes|
Improvements in transfection efficiency are possible with changes outside the scope of this protocol. For further guidance on obtaining better yields please contact us at firstname.lastname@example.org.
Subculture and expand cells to obtain culture with viability greater than 95% and viable cell density between 1.5 to 2.0 x 106 cells/mL at time of transfection.
If using a feed, booster, supplement, or enhancer, these can be added any time six hours post-transfection. Subcultures can also be prepared after six hours.
If using a GFP control, transfection efﬁciencies over 70% should be observed after 48 hours. For optimized procedures, efﬁciencies over 80% are reasonable.
Monitor expression levels and harvest upon observing plateaued titers. For typical processes, secreted proteins will be highest at 5 to 7 days post transfection.
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 is available by emailing us at email@example.com.
Gibco, Opti-Mem, FreeStyle, ExpiCHO, and ExpiCHO-S are trademarks or registered trademarks of Thermo Fisher Scientiﬁc. BalanCD is a FUJIFILM Irvine Scientiﬁc brand. HyClone, HyCell, TransFX-C are brands of Cytiva.