What are the morphological changes that I should be looking for in my 293FT cells as signs of lentivirus production?

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During lentivirus production, 293FT cells undergo the following morphological changes:
- They become multi-nucleated (syncytia development)
- They start to look like balloons or as if they are about to explode
- They often, but not always, lift off from the surface
- Untransfected 293FT cells leave empty spaces and “pile” up at other spots in the flask

Answer Id: E6403

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Do you recommend a specific FBS for culturing 293FT cells? Which plastic plates do you recommend?

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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.

Answer Id: E9317

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Why do I have to culture 293FT cells under Geneticin™ antibiotic selection?

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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.

Answer Id: E9318

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How do I know whether to choose lentivirus or adenovirus for viral expression?

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If you're interested in stable integration and selection, choose the lentiviral system. We offer both a Directional TOPO™ (D-TOPO™) and Gateway™ version of the kit to provide flexibility in the cloning of the gene of interest.

If you're looking for transient gene expression, choose the adenoviral system. We offer the Gateway™ cloning method for this product. It should be noted, however, that gene expression from both systems is typically detected within 24-48 hours of transduction, so both systems can be used for experiments of a transient nature. The main difference is that lentivirus integrates into the host genome and adenovirus does not. Higher viral titers are achieved with the adenovirus.

Answer Id: E4098

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What are syncytia?

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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.

Answer Id: E9319

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Why are lentiviruses able to transduce non-dividing cells when other viruses cannot?

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Lentiviruses gain entry into the host cell nucleus by means of an active import mechanism, using the host cell machinery. They enter the host cell nucleus and immediately integrate into the host cell genome. Hence, they are able to transduce both dividing and non-dividing cells. Retroviruses, on the other hand, use a passive entry mechanism and require a round of cell division before the virus can enter the host cell nucleus.

Answer Id: E6393

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What does the FT stand for in 293FT and why is this the most recommended producer cell line?

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The F stands for the high transfection efficiency of this particular 293 cell clone (called 293F) and the T stands for the SV40 large T antigen. If you want to use regular 293 cells or another 293T cell line, you will be able to produce virus, but the titers will be lower. The large T antigen expression plasmid is stably integrated in the 293FT cell and confers resistance to Geneticin™ antibiotic in these cells.

Answer Id: E4113

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Can the pLenti6/D-TOPO™ vector be used by itself as an expression vector (without packaging mix)?

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Yes, it will work as an expression vector by itself and can be stably selected with blasticidin. Please note that the vector will be about twice the size of most regular vectors. Therefore you may need to increase the amount of transfected vector to approximate molar equivalents.

Answer Id: E4117

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What are the advantages of the lentiviral system?

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The ViraPower™ Lentiviral System:
(1) effectively transduces both dividing and non-dividing cells
(2) efficiently delivers the gene of interest to mammalian cells in culture or in vivo
(3) produces a pseudotyped virus with a broadened host range
(4) includes multiple features designed to enhance the biosafety of the system

Answer Id: E4105

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What are the safety issues associated with the use of your viral systems?

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Both the lentiviral and adenoviral systems should be used following Biosafety Level 2 (BSL-2). We recommend strict adherence to all CDC guidelines for BSL-2 (as well as institutional guidelines). Thermo Fisher Scientific has also engineered specific safety features into the lentiviral system.

Consult the "Biosafety in Microbiological and Biomedical Laboratories" publication (www.cdc.gov, published by the CDC in the USA, describes BSL-2 handling) and the "Laboratory Biosafety Guidelines" publication (www.phac-aspc.gc.ca, published by the Centre for Emergency Preparedness and Response in Canada) for more information on safe handling of various organisms and the physical requirements for facilities that work with them.

Answer Id: E4099

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What is the difference between 293, 293H, 293F, and 293FT cells?

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The 293 cell line is a permanent line established from primary embryonal human kidney transformed with sheared human adenovirus type 5 DNA (Graham et al., 1977; Harrison et al., 1977). The E1A adenovirus gene is expressed in these cells and participates in transactivation of some viral promoters, allowing these cells to produce very high levels of protein.

The FreeStyle™ 293-F cell line is a clone of the 293 cell line and is intended for use with the FreeStyle™ 293 Expression System. These cells are adapted to suspension culture in FreeStyle™ 293 Expression Medium.

The 293H cell line was also cloned from 293 cells, and like 293F cells can be grown as either suspension or adherent cultures. Neither 293F or 293H attach very well in SFM. In serum-supplemented medium, both attach better, but 293H attaches better than 293F. 293H is also better for transient transfection. 293F cells have a faster doubling time than 293H, and are better suited for selection of stables and large scale cultures.

The 293FT Cell Line is derived from the 293F Cell Line and stably expresses the SV40 large T antigen from the pCMVSPORT6TAg.neo plasmid. Expression of the SV40 large T antigen is controlled by the human cytomegalo-virus (CMV) promoter and is high-level and constitutive.

Answer Id: E4493

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What precautions should I take with my 293FT cells to ensure high quality lentivirus production?

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• Use low passage 293FT cells. Do not use 293FT cells beyond passage 20. Freeze down many aliquots and grow for 2–4 passages prior to transfection.
• Passage cells in complete D-MEM containing G418 (500 µg/mL). Supplement the media with "non-essential" amino acids and sodium pyruvate (0.1 mM MEM Non-Essential amino acids and 1 mM MEM Sodium Pyruvate). Use Gibco™ FBS (Cat. No. 16000-044).
• Plate cells at a density of 5 x 10e6 per 100 mm dish. Cell density is very important. Make sure that the cells are growing well before re-plating prior to the day of transfection. Avoid overgrowth of 293FT cells when passaging.
• When plating for transfection the next day, do not add G418 to the media.

Answer Id: E6402

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Can I use HEK293 cells instead of 293FT cells for lentivirus production?

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Yes. You can use HEK293 cells for lentivirus production, but keep in mind that the titers will be lower. 293T cells and 293FT cells grow faster and are easier to transfect than HEK293 cells and result in higher titers of lentivirus. So most lentiviral protocols use 293FT cells to produce lentivirus. (It's not clear why the “large T” antigen is important for lentivirus production.)

Answer Id: E6383

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How much blasticidin do you usually put into culture medium to select for blasticidin-resistant clones for virus titration (HT1080 cells)?

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For HT1080 cells we typically use 10 μg/mL, but we strongly recommend that you generate a kill-curve for each antibiotic and cell line before proceeding. Most cell types respond to between 1 μg/mL and 10 μg/mL of blasticidin. For HT1080 cells, we typically use 100 μg/mL of Zeocin™ for Zeocin™-containing lentiviral vectors. But again, generation of a kill-curve is strongly suggested.

We strongly recommend titering on HT1080 cells to determine the absolute titer of infectious virus in your supernatant. The primary reason is that it's a way to standardize titers obtained in different labs. Transduction efficiency is high in these cells, and titering results are very accurate and reproducible, making HT1080 cells the gold standard for titering. You can then try different MOIs in other cell types based on HT1080 titers. For instance, you may require an MOI of 50 in one cell type or MOI of 10 in another cell type based on titers obtained in HT1080.Accurate titer, however, can be obtained in essentially any mammalian cell line, but 3T3 and HeLa cells have a lower transduction efficiency than HT1080 cells (for reasons unknown). Do not use 293FT cells for titering.

Answer Id: E4112

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Does the lentivirus produce any toxic viral genes?

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Lentiviruses produced with this system do not carry or express ANY viral genes and therefore have no associated toxicity issues. Only the protein expressed from the coding region between the LTR sites is incorporated into the mammalian cell chromosome and expressed. The lentivirus itself cannot replicate because of the built-in safety features.

Answer Id: E4116

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