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 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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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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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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Do 293FT cells lift off the plate during lentivirus production?

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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.

Answer Id: E9320

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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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Do you recommend a specific FBS for culture of the 293FT or 293A cells used in the ViraPower™ kits? What plastic plates do you recommend?

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We use mycoplasma-tested Gibco™ FBS (Cat. No. 16000-044) without any modifications. 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 293A and 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 (expect cell lysis in 293A cells when making adenovirus).

Answer Id: E4101

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What are the packaging limits for lentivirus and adenovirus? Can a 9 kb fragment be packaged into either?

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No, neither lentivirus nor adenovirus can take an insert as large as 9 Kb. Lentiviral packaging limits are around 6 kb and adenoviral packaging limits are around 7-7.5 kb. Above that, no virus is made.

For lentivirus, titers will generally decrease as the size of the insert increases. We have effectively packaged inserts of 5.2 kb with good titer (approx. 0.5 x 10^5 cfu/mL). The size of the wild-type HIV-1 genome is approximately 10 kb. Since the size of the elements required for expression from pLenti vectors add up to approximately 4-4.4 kb, the size of your gene of interest should theoretically not exceed 5.6-6 kb for efficient packaging (see below for packaging limits for individual vectors).
pLenti4/V5-DEST™ vector: 6 kb
pLenti6/V5-DEST™ vector: 6 kb
pLenti6/V5/D-TOPO™ vector: 6 kb
pLenti6/UbC/V5-DEST™ vector: 5.6 kb

For adenovirus, the maximum packagable size is approximately 7-7.5 Kb (see below for packaging limits for individual vectors).
pAd/CMV/V5-DEST™ vector: 6 kb
pAd/PL-DEST™ vector: 7.5 kb

Answer Id: E4095

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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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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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Will I get the same transduction efficiency with both lentivirus and adenovirus in the same cell line?

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This depends entirely on the target cell. Adenovirus requires the coxsackie-adenovirus receptor (CAR) and an integrin for efficient transduction. Lentivirus (with VSV-G) binds to a lipid in the plasma membrane (present on all cell types). With two totally different mechanisms of entry into the cell, there will always be differences in transduction efficiencies. However, the efficiency of transduction for both viral systems is easily modulated by the multiplicity of infection (MOI) used.

Answer Id: E4102

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What titers are typical with lentivirus?

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Titers between 1 x 10e5 and 3 x 10e5 cfu/mL (unconcentrated) are typical. If the titer is lower than 1x 10e5 cfu/mL, virus production was not optimal (arising for various reasons). Titers for the LacZ virus are typically in this low to mid 10e5 range. The sample lentiviral titer experiment shown in the ViraPower™ instruction manual shows lacZ lentivirus with a titer of 4.8 x 10e6 cfu/mL.

We strongly suggest that you titer your lentivirus on HT1080 cells, which allows you to compare titers from day-to-day within your lab and also with external 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.

Answer Id: E4109

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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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Why are 293FT cells cultured under Geneticin™ selection before transfection?

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For routine maintenance of 293FT cells, you need to add Geneticin™ (G418) antibiotic at a concentration of 500 μg/mL to maintain the Large T antigen plasmid/phenotype.

Answer Id: E4114

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How should I store lentivirus, adenovirus and viral vectors?

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Viral vectors:
Store lentiviral and adenoviral expression vectors at -20 degrees C. Due to their relatively large sizes, we do not recommend storing these vectors at -80 degrees C, as the vector solutions will completely freeze and too many freeze thaws from -80 degrees C will affect the cloning efficiency. At -20 degrees C, the vectors will be stable but will not freeze completely.

Virus:
Both adenovirus and lentivirus should be aliquoted immediately after production and stored at -80 degrees C.

Lentivirus is more sensitive to storage temperature and to freeze/thaw than adenovirus and should be handled with care. Adenovirus can typically be frozen/thawed up to 3 times without loss of titer, while lentivirus can lose up to 5% or more activity with each freeze/thaw. It is recommended to aliquot your virus into small working volumes immediately after production, freeze at -80 degrees C, and then thaw just one aliquot for titering. This way, every time you thaw a new aliquot it should be the same titer as your first tube.

Adenovirus can be kept overnight at 4 degrees C if necessary, but it is best to avoid this. Viruses will be most stable at -80 degrees C.

When stored properly, viral stocks should maintain consistent titer and be suitable for use for up to one year. After long-term storage, we recommend re-titering your viral stocks before use.

Answer Id: E4100

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