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View additional product information for ViraPower™ Adenoviral Promoterless Gateway™ Expression Kit - FAQs (K494000)
36 product FAQs found
In the single-step protocol for the BP/LR Clonase reaction, we would not recommend substituting the BP Clonase II/LR Clonase II enzymes with BP Clonase /LR Clonase enzymes as this would result in very low recombination efficiency.
Yes, we have come up with a single-step protocol for BP/LR Clonase reaction (http://www.thermofisher.com/us/en/home/life-science/cloning/gateway-cloning.html#1), where DNA fragments can be cloned into Destination vectors in a single step reaction, allowing you to save time and money.
We would recommend performing a BP reaction with a Donor vector in order to obtain an entry clone. This entry clone can then be used in an LR reaction with the Destination vector to obtain the new expression clone.
We do not offer the 5X LR Clonase buffer and 5X BP Clonase buffer as standalone products. They are available as part of the enzyme kits.
We do not offer any Gateway vectors for expression in plants.
Here are possible causes and solutions:
- Too much crude viral stock used:
-Reduce the amount of crude viral stock used for transduction or dilute the crude viral stock.
-Amplify the adenoviral stock.
-Concentrate the crude viral stock.
- Wild-type RCA (replication-competent adenovirus) contamination: Screen for RCA contamination. Plaque purify to isolate recombinant adenovirus or prepare a new adenoviral stock.
- Gene of interest is toxic to cells: Generation of constructs containing activated oncogenes or potentially harmful genes is not recommended.
Here are possible causes and solutions:
- Poor transduction efficiency due to:
-Mammalian cells not healthy: Make sure that your cells are healthy before transduction.
-Non-dividing cell type used: Transduce your adenoviral construct into cells using a higher MOI.
- MOI too low: Transduce your adenoviral construct into cells using a higher MOI.
- Low viral titer: Amplify the adenoviral stock using the procedure on page 20 of the manual (http://tools.thermofisher.com/content/sfs/manuals/virapower_adenoviral_system_man.pdf).
- Adenoviral stock contaminated with RCA (replication-competent adenovirus):
-Screen for RCA contamination.
-Prepare a new adenoviral stock or plaque purify to isolate recombinant adenovirus.
- Cells harvested too soon after transduction: Do not harvest cells until at least 24 hours after transduction.
- Cells harvested too long after transduction: For actively dividing cells, assay for maximal levels of recombinant protein expression within 5 days of transduction.
- Gene of interest is toxic to cells: Generation of constructs containing activated oncogenes or potentially harmful genes is not recommended.
Here are possible causes and solutions:
- Viral stocks stored incorrectly: Aliquot and store stocks at –80 degrees C. Do not freeze/thaw more than 10 times.
- Gene of interest contains a PacI site: Perform mutagenesis to change or remove the PacI site.
This could be due to insufficient dilution of the viral supernatant. We recommend titering the adenovirus stock using 10-fold serial dilutions ranging from 10e-4 to 10e-9.
Here are possible causes and solutions:
- Viral stocks stored incorrectly:
Aliquot and store stocks at –80 degrees C. Do not freeze/thaw more than 10 times.
- Incorrect titering of cell line used: Use the 293A cell line or any cell line with the characteristics discussed on page 23 of the manual http://tools.thermofisher.com/content/sfs/manuals/virapower_adenoviral_system_man.pdf).
- Agarose overlay incorrectly prepared: Make sure that the agarose is not too hot before addition to the cells; hot agarose will kill the cells.
- Viral stock with very low titer or very high titer: Titer adenovirus using a wider range of 10-fold serial dilutions (e.g.,10e2 to 10e8).
Here are possible causes and solutions:
- Low transfection efficiency due to:
-Shearing of adenoviral Destination vector DNA: Use care when handling the adenoviral Destination vector. Do not perform excessive manipulations (e.g.,vortexing or pipetting the solution vigorously) that may shear the DNA.
-Incomplete PacI digestion or digested DNA contaminated with phenol, ethanol, or salts: Repeat the Pac I digestion. Make sure purified DNA is not contaminated with phenol, ethanol, or salts.
-Unhealthy 293A cells; cells exhibit low viability: Use healthy 293A cells; do not overgrow cells.
-293A cells plated too sparsely on the day before transfection: Cells should be 90-95% confluent at the time of transfection.
-Plasmid DNA: transfection reagent ratio incorrect: Optimize such that plasmid DNA (in µg):Lipofectamine 2000 (in µL) ratio ranges from 1:2 to 1:3. If you are using another transfection reagent, optimize according to the manufacturer's recommendations.
- Viral supernatant too dilute: Concentrate virus using CsCl purification or any method of choice.
- Viral supernatant frozen and thawed multiple times: Do not freeze/thaw viral supernatant more than 10 times.
- Gene of interest is large: Viral titers generally decrease as the size of the insert increases; inserts larger than 6 kb (for pAd/CMV/V5-DEST) and 7.5 kb (for pAd/PL-DEST) are not recommended.
- Gene of interest is toxic to cells: Generation of constructs containing activated oncogenes or potentially harmful genes is not recommended.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Here are possible causes and solutions:
- LR reaction transformed into an E. coli strain containing the F' episome and the ccdA gene: Use an E. coli strain that does not contain the
F' episome for transformation (e.g.,TOP10, DH5α-T1R).
- Deletions (full or partial) of the ccdB gene from adenoviral Destination vector:
- The adenoviral Destination vectors are provided in solution and are ready to use in an LR reaction. However, if you wish to propagate them, we recommend using One Shot ccdB Survival2 T1R Chemically Competent Cells (Cat. No. A10460).
- Select for transformants in media containing 50-100 µg/mL ampicillin and 15-30 µg/mL chloramphenicol, to maintain the integrity of the vector.
- Prepare plasmid DNA from one or more colonies and verify the integrity of the vector before use.
Here are possible causes and solutions:
- Incorrect antibiotic used to select for transformants: Select for transformants on LB agar plates containing 100 µg/mL ampicillin.
- LR recombination reaction not treated with proteinase K: Treat reaction with proteinase K before transformation.
- Too much entry clone DNA used in the LR reaction: Use 50-150 ng of the entry clone in the LR reaction.
- Inappropriate ratio of entry clone:DEST vector used in the LR reaction: Aim for a 1:1 molar ratio of entry clone:DEST vector.
- LR recombination of >5 kb insert only incubated for 1 hr: For inserts larger than 5 kb, we recommend to incubate the LR reaction overnight. Note: This overnight incubation will also boost colony count for smaller inserts.
- Adenoviral Destination vector DNA was sheared: Use care when handling the adenoviral Destination vector. Do not perform excessive manipulations (e.g.,vortexing or pipetting the solution vigorously) that may shear the DNA.
- Didn't use the suggested amount of LR Clonase II enzyme mix or LR Clonase II enzyme mix was inactive:
-Make sure to store the LR Clonase II enzyme mix at –20 degrees C.
-Do not freeze/thaw the LR Clonase II enzyme mix more than 10 times.
-Use the recommended amount of LR Clonase II enzyme mix (see page 14 of the manual [http://tools.thermofisher.com/content/sfs/manuals/pad_dest_man.pdf]).
-Test another aliquot of the LR Clonase II enzyme mix.
- Not enough LR reaction transformed: Transform 2-3 µL of the LR reaction into the appropriate competent E. coli strain. Use E. coli cells with a transformation efficiency >1 x 10e8 cfu/µg.
- Not enough transformation mixture plated: Increase the amount of E. coli plated.
The ViraPower Adenoviral Expression System includes the following features designed to enhance its biosafety:
- The entire E1 gene is deleted in the adenoviral expression vectors (pAd/CMV/V5-DEST and pAd/PLDEST) and supplied in trans in the 293A producer cell line. Since expression of E1 (E1a and E1b) proteins is required for the expression of the other adenoviral viral genes (e.g., late genes), adenovirus produced using this system is replication-incompetent in any mammalian cells that do not express the E1a and E1b proteins.
- The E3 gene is completely dispensable for in vitro applications and hence is deleted as well from the adenoviral expression vector backbone.
- The adenovirus does not integrate into the host genome upon transduction. Because the virus is replication-incompetent, the presence of the viral genome is transient and will eventually be diluted out as cell division occurs.
Despite the presence of the above safety features, the adenovirus produced can still pose some biohazardous risk since it can transduce primary human cells. For this reason, we highly recommend that you treat adenoviral stocks generated using this system as Biosafety Level 2 (BL-2) organisms and strictly follow all published guidelines for BL-2. Furthermore, exercise extra caution when creating adenovirus carrying potential harmful or toxic genes (e.g., activated oncogenes) or when producing large-scale preparations of virus (see page 10 of the manual [http://tools.thermofisher.com/content/sfs/manuals/virapower_adenoviral_system_man.pdf]).
For more information about the BL-2 guidelines and adenovirus handling, refer to the document, “Biosafety in Microbiological and Biomedical Laboratories,” 4th Edition, published by the Centers for Disease Control (CDC) (www.cdc.gov/biosafety/publications/index.htm).
Getting a cell transfected and observing productive viral transduction are two different things. If only one or two cells in your lawn are producing virus, it will take quite a while for that to be visible to the naked eye (longer than most are willing to wait). Transfection efficiency is correlated with virus production because the more cells you get DNA into, the higher chance you have of seeing virus production within the first week or two. If your transfection efficiency is low, you will eventually see virus being produced, but you have to wait a long time to see it.
The pAd/CMV/V5-DEST or pAd/PL-DEST adenoviral constructs do not integrate into the host genome. Once transduced into the mammalian cell of interest, your recombinant protein is expressed as long as the viral genome is present. For actively dividing cells, transgene expression decreases over time and can be down to background levels within 2 weeks after transduction. In non-dividing cells such as quiescent CD34+ cells or animal tissues (skeletal muscle, neurons, liver), transgene expression is more stable and can persist for as long as 6 months post-transduction.
In actively dividing cells (doubling time of every 24 hours), we have found that transgene expression is generally detectable within 24 hours of transduction, with maximal expression observed at 48-96 hours (2-4 days) post-transduction. Expression levels generally start to decline after 5 days post-transduction. In cell lines that exhibit longer doubling times or in non-dividing cell lines, high levels of transgene expression persist for a longer period of time.
Human adenovirus type 5 (Ad5) enters target cells via the coxsackie virus and adenovirus receptor (CAR), followed by an integrin-mediated internalization mechanism. CAR/integrin proteins are ubiquitously present on mammalian cells, thus affording adenovirus the ability to transduce a very broad range of cell types. If your specific cell type has very low expression of CAR, adenoviral transduction will be inefficient, in which case you may need to use a very high MOI (in the 100s) to get good expression.
The backbone for our ViraPower adenoviral expression vectors is human adenovirus type 5 (Ad5). Ad5 entry into cells is achieved by binding to the coxsackie virus and adenovirus receptor (CAR), followed by an integrin-mediated internalization mechanism. For target cells that have sufficient expression of the CAR receptor and are actively dividing, it should be possible to get adenovirus transduction efficiencies in the range of 80-90%, as long as an adequate MOI is used.
Note: There is variability in the transduction efficiencies of different cell types. Example: In HT1080 cells, which are readily transduced with adenovirus, transduction efficiencies are around 90% with an MOI of 1. In some cell types, you may need to use a 10-fold higher MOI to get the same transduction efficiency.
Crude adenovirus titers are generally 1 x 10e7 to 1 x 10e8 plaque forming unts (pfu)/mL. You can use this stock to infect a new batch of 293A cells to generate a higher-titer viral stock (i.e., amplify the virus). Amplification allows production of a viral stock with a titer ranging from 1 x 10e8 to 1 x 10e9 pfu/mL. Adenovirus can be concentrated to titers as high as 1 x 10e11 pfu/mL using a variety of methods (e.g., CsCl purification).
Adenovirus can be concentrated to titers as high as 1 x 10e11 pfu/mL using a variety of methods (e.g., CsCl purification; please find a reference on page 25 of the manual [http://tools.thermofisher.com/content/sfs/manuals/virapower_adenoviral_system_man.pdf]).
Once you have created a crude viral stock, you can use this stock to infect a new batch of 293A cells to generate a higher-titer viral stock (i.e., amplify the virus). The titer of the initial viral stock obtained from transfecting 293A cells generally ranges from 1 x 10e7 to 1 x 10e8 plaque forming units (pfu)/mL. Amplification allows production of a viral stock with a titer ranging from 1 x 10e8 to 1 x 10e9 pfu/mL and is generally recommended. Please refer to page 19 in the manual (http://tools.thermofisher.com/content/sfs/manuals/virapower_adenoviral_system_man.pdf) for specific instructions for amplification.
Note: Other 293 cell lines or cell lines expressing the E1 proteins are suitable for amplification.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
We recommend aliquoting adenoviral stocks immediately after production into small working volumes, and storing at –80 degrees C for long-term storage. Since adenovirus is non-enveloped, viral stocks remain relatively stable and some freezing and thawing of the viral stocks is acceptable. We do not recommend freezing and thawing viral stocks more than 10 times, as loss of viral titer can occur. When stored properly, viral stocks of an appropriate titer should be suitable for use for up to one year. After long-term storage, we recommend re-titering your viral stocks before use.
Most of the adenovirus is contained within the floating cells and is not released into the medium until those cells burst. We recommend changing the medium every 3 days or so until it is obvious that a lot of cells become big and rounded and are detaching from the plastic. Once a cell bursts, the free viruses rapidly infect the neighboring cells. If you're ever worried that you're losing infected cells (and therefore potential virus) in your medium changes, you can always save the medium with the floating cells, freeze/thaw it 3 times and then use a little (maybe 1/10th) and add it back to your culture with fresh media. Or, replace only half of the medium with fresh medium and do this more often than every three days.
Any 293-derived cell line or other cell line that expresses the E1 proteins may be used to produce adenovirus. In 293A cells (recommended for adenovirus production), "A" stands for "adherent" because the 293A cells (which are just a single-cell clone of regular 293) tend to adhere and form nice flat monolayers in tissue culture dishes. This is why they work so well for plaque assays. Regular 293 cells will not form the same type of monolayers; they exhibit holes and gaps during growth.
We use mycoplasma-tested Gibco FBS (Cat. No. 16000-044) and use the following plasticware for 293A cells:
T175: Fisher Cat. No. 10-126-13; this is a Falcon flask with a 0.2 µm vented plug seal cap.
T75: Fisher Cat. No. 07-200-68; this is a Costar flask with a 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).
The size of the wild-type adenovirus type 5 genome is approximately 35.9 kb. Studies have demonstrated that recombinant adenovirus can efficiently package up to 108% of the wild-type virus size from E1- and E3-deleted vectors. Taking into account the size of the elements required for expression from each adenoviral destination vector, make sure that your DNA sequence or gene of interest does not exceed the size indicated for efficient packaging (see below for packaging limits for individual vectors):
pAd/CMV/V5-DEST: 6 kb
pAd/PL-DEST: 7.5 kb
You can transfect your adenoviral construct into your expression cell line (or the 293A cells) to see if the protein will be expressed without waiting the two weeks it takes to make virus. Transfection efficiency will be low due to the large size of the plasmid, so it may require adding more lipid-DNA complexes to the medium than indicated in the ViraPower Adenoviral Expression System manual. The adenoviral construct should not be digested with Pac I when doing this, as supercoiled plasmids transfect more efficiently. If checking expression in 293A cells, harvest 2-3 days post-transfection.
Before you can transfect your expression clone into 293A cells, you must expose the left and right viral inverted terminal repeats (ITRs) on the vector to allow proper viral replication and packaging. This also removes bacterial sequences (i.e., pUC origin and ampicillin resistance gene). Both pAd/CMV/V5-DEST and pAd/PL-DEST ;vectors contain Pac I restriction sites (see maps on pages 20 and 22 of the manual (http://tools.thermofisher.com/content/sfs/manuals/pad_dest_man.pdf), respectively, for the location of the Pac I sites).
Note: Make sure that your DNA sequence of interest does not contain any Pac I restriction sites. If you are unable to use the Pac I site, you can use the Swa I site.
Once you have generated your pAd/CMV/V5-DEST or pAd/PL-DEST expression clone, you may use any method of choice to prepare purified plasmid DNA. We recommend isolating plasmid DNA using the PureLink HiPure Plasmid Midiprep Kit (Cat. No. K210004) or CsCl gradient centrifugation.
Note: We recommend performing restriction analysis to verify the integrity of your expression construct after plasmid preparation.
We recommend storing adenoviral expression vectors at –20 degrees C. Due to their relatively large size, we do not recommend storing these vectors at –80 degrees C, as the vector solution will completely freeze and too many freeze thaws from –80 degrees C will affect the cloning efficiency.
The pAd-DEST plasmids are large (>34 kb in size) and excessive manipulations can shear the DNA, resulting in reduced LR recombination efficiency. When working with pAd-DEST plasmids, do not vortex or pipet the solution vigorously. These vectors are supplied supercoiled, as lyophilization methods and room temperature storage may result in plasmid damage. Freeze thaws are acceptable as long as shearing is prevented.
No, our system uses human adenovirus type 5 (Ad5).
The backbone for our ViraPoweradenoviral expression vectors is human adenovirus type 5 (Ad5).
If you are 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 are looking for transient gene expression, choose the adenoviral system. We offer the Gateway cloning method for this product. Adenoviral vectors can be amplified several times in 293A cells, whereas the only method to concentrate lentivirus is by centrifugation. Adenovirus requires that host cells have th CAR receptor for efficient transduction, whereas due to the VSVG membrane coat on lentivirus particles, these viruses have broad tropism for a variety of mammalian cell types.
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.
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.
Adenoviral expression is used for transient expression, whereas lentiviral expression is used for longer-term expression. Adenoviral vectors can be amplified several times in 293A cells, whereas the only method to concentrate lentivirus is by centrifugation. Adenovirus requires that host cells have the CAR receptor for efficient transduction, whereas due to the VSVG membrane coat on lentivirus particles, these viruses have broad tropism for a variety of mammalian cell types.