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View additional product information for pAd/CMV/V5-DEST™ Gateway™ Vector Kit - FAQs (V49320)
22 product FAQs found
Adenovirus: For cells that have sufficient expression of the CAR receptors 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 (for instance in HT1080 cells, which are readily transducible with adenovirus, transduction efficiencies are around 90% with an MOI of 1).
Lentivirus: Similar transduction efficiencies are possible with lentivirus in certain cell types (for instance, in HT1080 cells, which are readily transducible with lentivirus as well as adenovirus, an MOI of 1 gives transduction efficiencies of around 90%). There is definitely variability in the transduction efficiencies, based on cell type, for both adenovirus and lentivirus. For instance, in some cell types, you may need to use a 10-fold higher MOI to get the same transduction efficiency.
No. The recombinant viral genome does not integrate into the host genome, nor does it have any mechanism of replication. However, the presence of the adenovirus terminal protein (TP) covalently linked to the ends of the viral DNA makes the viral genome very stable in the nucleus.
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 is low due to the large size of the plasmid, so it may require adding more complexes to the media than indicated in the manual (the ViraPower kit manual actually recommends less than the Lipofectamine 2000 reagent manual). The plasmid 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 posttransfection.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Although we have not constructed these in house, to get a true empty adenovirus, take our adenoviral destination vector and recombine it with an empty entry vector. This will replace the Gateway destination cassette (ccdB, CmR, etc.) with just a short MCS. Alternatively, you could use the adenoviral destination vector directly to make virus without a GOI. This isn't exactly an "empty" vector, but it should give no mammalian expression.
Typically the lacZ control works very well, but there really should be no obvious cell lysis for 9-12 days post transfection (viral life cycle is about 3 days, but the transfections are usually low efficiency and you typically need ~3 rounds of virus replication before you see cell lysis on a plate-wide scale, sometimes a bit longer). Once you have virus in your hands, however, and apply the virus to 293 cells you should see lysis in just 3 days. Crude viral titers typically are greater than 10e9 pfu/mL.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
You should expect to see many plaques from a single 6-well transfection with 2 µg PacI-digested DNA. Most of the time, we see several areas in the well that are showing CPE (cytopathic effect) resulting from virus replication. This is when we do our final media change because 3-4 days after these discrete areas are visible, your whole well will look this way and will be ready to harvest.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Getting a cell transfected and observing productive viral infection 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 more 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.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
You can obtain a titer of 10e12 pfu/mL (after concentration). The unconcentrated adenovirus titer should be at least 1x 10e8 pfu/mL.
We generally produce adenoviral stocks in 293A cells using the following optimized transfection conditions. The amount of adenovirus produced using these recommended conditions is approximately 10 mL of crude viral lysate with a titer ranging from 1 x 10e7 to 10e8 pfu/mL. We use Lipofectamine 2000 reagent for transfection. If using another transfection reagent, use the manufacturer's recommendations.
-Tissue culture plate size: 6-well plate (one well per adenoviral construct)
-Number of 293A cells to transfect: 5 x 10e5 cells
-Amount of Pac I-digested pAd/PL-DEST expression plasmid: 2 µg
-Amount of Lipofectamine 2000: 6 µl
-293A cells should be plated 24 hours prior to transfection in complete medium, and should be 90-95% confluent on the day of transfection. Make sure the cells are healthy at the time of plating.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
(1) Easiest and fastest method to generate adenovirus clones because the 100% efficiency of the Gateway system compared to traditional, less-efficient methods.
(2) Can perform protocols that are not possible in other adeno systems because of Gateway Technology.
(3) Ability to grow to high titer: 10e8 pfu/mL in crude preparations and 10e11-10e12 pfu/mL concentrated.
(4) Since the adenovirus is non-enveloped, it is very stable in cell culture media and can be freeze-thawed with little loss in activity. The adenovirus can undergo three freeze/thaw cycles without any significant loss of activity.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Adenovirus enters target cells via the coxsackie-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 cell has little-to-no CAR, then adenoviral transduction will be inefficient; in which case you may need very high MOIs (in the 100s) to get good expression.
No. The ViraPower system uses adenovirus type 5. Adenoviruses (Adenoviridae) and adeno-associated viruses (Parvoviridae) are completely different. Adeno-associated viruses are often associated with adenovirus infections, hence the name. Since they are thought to be virtually non-pathogenic, they are attractive vectors for gene therapy. The disadvantage is that they can package only about half the foreign DNA that adenoviruses can.
Clone your gene of interest into the pAd/CMV/V5-DEST (or pAd-PL-DEST if you want to use your own promoter). Prior to cloning, if desired, propagate this vector in One Shot ccdB Survival 2 T1R Competent Cells (Cat. No. A10460) as described below. After cloning your gene of interest, propagate in E. coli strain TOP10. pAd/CMV/V5-GW/lacZ is provided as a positive control vector for expression.
Digest recombinant plasmid with Pac I to expose the ITRs (inverted terminal repeats).
Transfect (we recommend Lipofectamine 2000 reagent) E1-containing cells (293A cells) with linear DNA (only 10% of transfected cells will make virus).
Infected cells will ball up, and release virus to surrounding cells, which in turn will be killed and ball up. Look for plaques in the monolayer created by areas cleared by detaching, balled up cells (it takes 8-10 days to see visible plaques from this initial transfection).
Collect a crude viral lysate.
Amplify the adenovirus by infecting 293A producer cells with the crude viral lysate. Harvest virus after 2-3 days when cells ball up. Determine the titer of the adenoviral stock by performing a plaque assay. The virus generated is adenovirus type 5 (subclass C).
Add the viral supernatant to your mammalian cell line of interest to transduce cells.
Assay for recombinant protein of interest.
Once you have your gene of interest in the adenoviral vector, you can simply re-amplify when you need more of the virus. You do not need to repeat cloning steps and transfections each time.
When cloning or propagating DNA with unstable inserts (such as lentiviral DNA containing direct repeats), we recommend using the following modifications to reduce the chance of recombination between direct repeats:
- Select and culture transformants at 25-30 degrees C.
- Do not use "rich" bacterial media as they tend to give rise to a greater number of unwanted recombinants.
-If your plasmid confers chloramphenicol resistance, select and culture transformants using LB medium containing 15-30 µg/mL chloramphenicol in addition to the antibiotic appropriate for selection of your plasmid.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
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.
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.
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).
Viral vectors:
Store lentiviral and adenoviral expression vectors (plasmid DNA) 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. Glycerol stocks of vectors transformed into bacteria should always be stored at -80 degrees C.
Virus:
Both adenovirus and lentivirus particles 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 particles 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.
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.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
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
While the F' plasmid does contain the ccdA gene that can inhibit or reduce the toxicity of the ccdB gene product, the ccdA expression level is likely to be too low, or inhibition may not be complete, and the bacteria would still be exposed to the ccdB gene product and thus not grow. Therefore, bacterial strains containing the F' plasmid are not recommended as hosts for propagation of ccdB containing vectors.
For propagation of Gateway vectors containing ccdB, we recommend the One Shot ccdB Survival 2 T1R Competent Cells (A10460), which were specifically designed for that purpose. However, please note that these cells are not validated for propagation of other ccdB-containing vectors like the older pZErO plasmids, and in most cases they are not expected to work due to very high levels of ccdB protein expressed in those vectors.
In addition to the CAAT and TATA boxes, the CMV promoter in pcDNA3.1 vector contains sequence homologous to base pairs 137 to 724 of the sequence submitted by Boshart, et al (GenBank Accession # K03104). The complete enhancer region is contained between 214 and 620 of this sequence. Therefore, by this definition, the CMV promoter could be said to be "complete".
Please note that this promoter does not contain an intron. Some people believe that the complete promoter must contain the intron, but that has not been demonstrated to be necessary for expression.