Search
Search
查看更多产品信息 ViraPower™ Adenoviral Promoterless Gateway™ Expression Kit - FAQs (K494000)
11 个常见问题解答
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.