ViraPower™ HiPerform™ Promoterless Gateway™ Vector Kit

If 293FT cells detach shortly after transfection (4 hours to overnight):

- This may be a sign of Lipofectamine 2000 toxicity. Cells may have been plated too sparsely prior to transfection.
- The cells may not have been handled gently enough (these cells have a tendency to lift off easily).
- The cells may have been kept at room temperature for too long.

If cells detach 48 to 72 hours post-transfection:
- If the cells lift off in large sheets, this may be a sign of lentivirus production.

Syncytia are large multi-nucleated cells that result from VSV-G-induced fusion with neighboring 293FT producer cells. Syncytia production is indicative of high transfection efficiency and lentivirus production. Keep in mind, though, that the absence of syncytia does not mean that virus will not be produced.

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The 293FT cell line stably expresses the SV40 large T antigen from the pCMVSPORT6Tag.neo plasmid that contains the neomycin resistance marker. In order to maintain the plasmid/phenotype, the cells have to be routinely cultured in medium containing Geneticin (G418) antibiotic at a concentration of 500 µg/mL.

We use Mycoplasma-tested Gibco FBS (Cat. No. 16000-044). We have observed that when 293FT cells are cultured in the presence of this FBS following the instructions in the manual, virus production is better than that obtained with many other serum sources. We use the following plasticware for 293FT cells:

T175-Fisher Cat. No. 10-126-13; this is a Falcon flask with 0.2 µm vented plug seal cap.
T75-Fisher Cat. No. 07-200-68; this is a Costar flask with 0.2 µm vented seal cap.
100 mm plate-Fisher Cat. No. 08-772E; this is a Falcon tissue culture-treated polystyrene plate.
We get excellent adherence on these plates under routine cell culture/maintenance conditions.

We offer the ViraPower HiPerform Promoterless Gateway Expression System (Cat. No. A11145) that combines ViraPower HiPerform Lentiviral and MultiSite Gateway technologies to facilitate easy recombination-based cloning and lentiviral-based high-level expression of a target gene from any promoter of choice in dividing and non-dividing mammalian cells. The ViraPower HiPerform Promoterless GatewayVector Kit is available separately (Cat. No. A11146).