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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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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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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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 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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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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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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Once I make lentivirus, can I amplify the virus or do I need to do another transfection?

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The lentiviruses produced in this system will not replicate under any conditions. You must perform a fresh transfection each time you need more virus.

Answer Id: E4118

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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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How do I concentrate the lentiviral stock?

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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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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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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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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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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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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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