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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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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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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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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How does the lentiviral system work? How do I make the lentivirus?

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Clone your gene of interest into one of our lentiviral expression vectors. We have a Directional TOPO™ version (pLenti6/V5/D-TOPO™) and a Gateway™ version (pLenti6/V5-DEST™ vector). Co-transfect your recombinant vector along with the optimized ViraPower™ packaging mix into the 293FT producer cell line using Lipofectamine™ 2000 reagent (if using a different transfection reagent, follow the manufacturer's recommendations). Harvest the viral supernatant and determine the titer of the virus. Add the viral supernatant to your mammalian cell line of interest at the appropriate MOI. Assay for "transient" expression of your recombinant protein or select for stably transduced cells using the appropriate selection antibiotic, if desired, then examine expression of your protein of interest.

Answer Id: E4104

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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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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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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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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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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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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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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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