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Product FAQ

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

Answer

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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Product FAQ

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

Answer

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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Product FAQ

How should I store lentivirus, adenovirus and viral vectors?

Answer

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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Product FAQ

How do I concentrate the lentiviral stock?

Answer

Ultracentrifugation is the most commonly used approach and is typically very successful (see Burns et al. (1993) Proc Natl Acad Sci USA 90:8033-8037; Reiser (2000) Gene Ther 7:910-913). Others have used PEG precipitation. Some purification methods are covered by patents issued to the University of California and Chiron.

Adenovirus is concentrated using CsCl density gradient centrifugation (there is a reference for this procedure in our adenovirus manual) or commercially available columns.

Answer Id: E4110

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Product FAQ

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

Answer

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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Product FAQ

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

Answer

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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Product FAQ

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?

Answer

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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Product FAQ

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

Answer

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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Product FAQ

What are the advantages of the lentiviral system?

Answer

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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Product FAQ

Can I remove the CMV promoter from the pLenti6/V5-D-TOPO™ or pLenti6/V5-DEST™ vectors?

Answer

Yes, you can use restriction enzymes Cla I (cutting at 1796) and BamH I (cutting at 2401) to remove the CMV promoter from the pLent6/V5-D-TOPO™ vector. Use Cla I and Spe I for the pLenti6/V5-DEST™ vector. Alternatively, we offer promoter-less lentiviral vectors that do not contain a promoter.

Answer Id: E4111

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Product FAQ

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

Answer

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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Product FAQ

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

Answer

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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Product FAQ

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

Answer

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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Product FAQ

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

Answer

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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Product FAQ

What are syncytia?

Answer

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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