What is the difference between 293, 293H, 293F, and 293FT cells?
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: 4493
What are the packaging limits for lentivirus and adenovirus? Can a 9 kb fragment be packaged into either?
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: 4095
How do I know whether to choose lentivirus or adenovirus for viral expression?
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: 4098
What are the safety issues associated with the use of your viral systems?
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). Life Technologies 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: 4099
How should I store lentivirus, adenovirus and 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.
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: 4100
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?
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: 4101
Will I get the same transduction efficiency with both lentivirus and adenovirus in the same cell line?
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: 4102
What is MOI, and how do I know which MOI to use?
MOI stands for multiplicity of infection. Theoretically, an MOI of 1 will provide 1 virus particle for each cell on a plate, while an MOI of 10 represents ten virus particles per cell. However, several factors can influence the optimal MOI including the nature of your mammalian cell line, (non-dividing vs. dividing), transduction efficiency, your application of interest, and your protein of interest.
When transducing your adenoviral or lentiviral construct into the mammalian cell line of choice for the first time, we recommend using a range of MOIs (0. 0.5, 1, 2, 5, 10, 50) to determine the MOI required to obtain optimal gene expression (MOIs greater than 50 (such as MOI 100) are common for the transduction of neurons with lentivirus). After you determine the MOI that gives optimal gene expression, subsequent transductions can be performed at the optimal MOI.
Answer Id: 4103
How does the lentiviral system work? How do I make the lentivirus?
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: 4104
What are the advantages of the lentiviral system?
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: 4105
What type of virus is the lentivirus?
The lentivirus is a retrovirus and is loosely based on HIV-1, however it has been altered to function solely as a gene delivery vehicle without subsequent viral replication or disease. Specific HIV-1 genes have been deleted to enhance safety. The HIV-1 genes are only expressed in the producer cells (293FT) and none of them are packaged into the viral genome and thus are never expressed in the transduced target cell.
Answer Id: 4106
Can I transduce my specific cell type with a lentivirus? How does lentivirus infect cells?
The lentiviral envelope contains (is pseudotyped with) the vesicular stomatitis virus glycoprotein (VSV-G), which allows the lenitivirus to interact with its target cell in a receptor-independent manner. This receptor-independent entry into the target cell likely involves endocytosis (see Espenshade et al (2002) Proc Natl Acad Sci USA 99:11694; Aiken (1997) J Virol 71:5871). Since the interaction of this pseudotyped lentivirus with mammalian cells is receptor-independent, the lentivirus should be able to (in theory) transduce any mammalian cell type.
Theodore Friedmann was the pioneer in the field of lentiviral host range and has a tremendous number of publications and reviews describing VSV-G pseudotyping of retroviruses and their tropisms:
VSV-G binding to target cells is receptor-independent. It interacts with a common phospholipid (phosphatidylserine has been proposed to be the most probable target) in the target cell membrane and this is how it confers such broad tropism: mammalian cells, fish, etc. (Burns et al (1993) Proc Natl Acad Sci USA 90:8033).
One group has shown that VSV-G pseudotyped retroviruses can transduce amoebas (see Que et. al. Mol. Biochem. Parasitol. (1999) 99:237-45).
The VSV-G element was derived from the San Juan strain of the Indiana serotype (Rose and Gallione (1981) J Virol 39:519), as referenced by Ory et al (1996) PNAS 93:11400).
Answer Id: 4107
How do I choose between the Gateway® and TOPO® versions of the lentiviral systems?
The TOPO® Cloning method is an easy-to-use, 5-minute benchtop PCR cloning method, and we have developed many kits based on this technology. You may want to choose this method if you have only one construct to make. If you plan to place your gene of interest into several different expression systems, you may want to consider the Gateway® Cloning Technology, also used in many of our expression kits.
Answer Id: 4108
What titers are typical with lentivirus?
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: 4109
How do I concentrate the lentiviral stock?
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: 4110