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This is intended as a guide only; for full experimental details please read the references provided.
Protocol for Transient Gene Expression in CHO 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™ F17 Expression Medium
Gibco™ ExpiCHO™ Expression Medium (with ExpiCHO-S™ Cells)
|Cytiva||HyClone HyCell TransFX-C|
|FUJIFILM Irvine Scientific||BalanCD CHO (Recommended)|
Cell densities used are for typical CHO 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, ExpiCHO™ media and ExpiCHO-S™ cells can support viable cell densities over ~12 x 106 cells/mL instead of ~4.0 x 106 cells/mL. Accordingly, this system should be transfected at 3.15 x 106 cells/mL instead of 1.05 x 106 cells/mL.
The typical pDNA and PEI STAR™ concentrations (1.0 and 5.0 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 recommend co-varying these parameters over the corresponding ranges to ﬁnd the optimal conditions:
|PEI Concentration||3.50 to 6.50 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.
Depending on the cell culture media, it may be necessary to add sodium butyrate to the media to obtain gene expression in CHO suspensions. To determine if necessary, prepare 5 post-transfection subcultures and add sodium butyrate to ﬁnal concentrations of 0, 2.5, 5.0, 7.5, or 10 mM to ﬁnd appropriate concentration based on the ﬁnal yields. Sodium valproate can be used instead.
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. Other processes, such as using low temperatures and/or using a batch feed to obtain maximal yields, will change the peak harvest window.
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.