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View additional product information for BLOCK-iT™ Adenoviral RNAi Expression System - FAQs (K494100)
27 product FAQs found
Yes. The miR miRNA vectors are Gateway cloning compatible, and you could use Gateway cloning to transfer the miR miRNA expression cassette to any of our Gateway-adapted viral expression vectors.
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
Please ensure that the recommended filter sets for detection of fluorescence are used. Use an inverted fluorescence microscope for analysis. If desired, allow the protein expression to continue for 1-3 days before assaying for fluorescence.
The target sequence used may contain strong homology to other genes; please select a different target region.
Perform a kill curve to determine the antibiotic sensitivity of your cell line. Ensure that viral stocks are stored properly at -80 degrees C, and do not undergo freeze/thaw more than 3 times. Lastly, transducer the lentiviral contruct into cells in the presence of Polybrene reagent.
Ensure that the competent cells used were stored properly at -80 degrees C, and thawed on ice for immediate use. When adding DNA, mix competent cells gently: do not mix by pipetting up and down. Also do not exceed the maximum recommended amount of DNA for transformation (100 ng) or allow the volume of DNA added to exceed 10% of the volume of the competent cells, as these may inhibit the transformation.
Please check to ensure that your medium containing fetal bovine serum (FBS) is reduced in tetracycline. Many lots of FBS contain tetracycline, as FBS is often isolated from cows that have been fed a diet containing tetracycline, leading to low basal expression of shRNA. Ensure that a cell line expressing the Tet repressor is being used, and that the cells used are transduced at a suitable MOI. If creating your own Tet repressor-expressing cell line, wait at least 24 hours before transducing cells with your shRNA construct.
Low expression levels can be due to several factors. Please see the suggestions below:
- Low transfection efficiency: ensure that antibiotics are not added to the media during transfection, and that cells are at the proper cell confluency; optimize transfection conditions by varying the amount of transfection reagent used.
- Try a time course assay to determine the point at which the highest degree of gene knockdown occurs.
- Mutations are present in your construct: analyze the transformants by sequencing the ds oligo insert to verify its sequence.
- Target region is not optimal: select a different target region.
- Ensure siRNA is designed according to guidelines listed in the respective manual.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
You can try to scale back the amount of transfection reagent used, or use a different reagent for the transfection. Additionally, ensure that the plasmid used is pure and properly prepared for transfection.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
Difficulties sequencing could occur because the hairpin sequence is an inverted repeat that can form secondary structure during sequencing, resulting in a drop in the sequencing signal when entering the hairpin. If you encounter difficulties while sequencing, please try the following:
- Use high-quality, purified plasmid DNA for sequencing. We recommend preparing DNA using the Invitrogen PureLink HQ Mini Plasmid Purification Kit (Cat. No. K2100-01) or S.N.A.P. Plasmid DNA MidiPrep Kit (Cat. No. K1910-01).
- Add DMSO to the sequencing reaction to a final concentration of 5%.
- Increase the amount of template used in the reaction (up to twice the normal concentration).
- Standard sequencing kits typically use dITP in place of dGTP to reduce G:C compression. Other kits containing dGTP are available for sequencing G-rich and GT-rich templates. If you are using a standard commercial sequencing kit containing dITP, obtain a sequencing kit containing dGTP (e.g., dGTP BigDye Terminator v3.0 Ready Reaction Cycle Sequencing Kit, Cat. No. 4390229) and use a 7:1 molar ratio of dITP:dGTP in your sequencing reaction.
We highly recommend sequencing positive transformants to confirm the sequence of the ds oligo insert. When screening transformants, we find that up to 20% of the clones may contain mutated inserts (generally 1 or 2 bp deletions within the ds oligo). The reason for this is not known, but may be due to triggering of repair mechanisms within E. coli as a result of the inverted repeat sequence within the ds oligo insert. Note: Entry clones containing mutated ds oligo inserts generally elicit a poor RNAi response in mammalian cells. Identify entry clones with the correct ds oligo sequence and use these clones for your RNAi analysis.
Mutated inserts could also be caused by using poor-quality single-stranded oligos. Use mass spectrometry to check for peaks of the wrong mass, or order HPLC- or PAGE-purified oligos to avoid this problem.
- Verify that the sequence of the bottom-strand oligo is complementary to the sequence of the top-strand oligo.
- For the shRNA vectors, make sure that you mix single-stranded oligos with complementary sequences. The top-strand oligo should include CACC on the 5' end, while the bottom-strand oligo should include AAAA on the 5' end.
- For the miRNA vectors, make sure that the top-strand oligo includes TGCT at the 5' end and that the bottom-strand oligo includes CCTG at the 5' end.
Please review the possibilities below:
- Single-stranded oligos designed incorrectly; verify that the sequence of the bottom-strand oligo is complementary to the sequence of the top strand oligo.
- Ensure that oligos are annealed at room temp for 5-10 minutes after heating to 95 degrees C.
- Check the molar ratio you are using for annealing top and bottom-strand oligo; equal amounts should be used.
This is not necessary, as the transfer preserves the orientation of the cassette. However, if you wish to sequence your DEST expression construct, we recommend the following primers:
Primer Sequence:
pAd forward priming site: 5'-GACTTTGACCGTTTACGTGGAGAC-3'
pAd reverse priming site: 5'-CCTTAAGCCACGCCCACACATTTC-3'
We recommend using One Shot ccdB Survival T1R chemically competent cells (Cat. No. C751003) for transformation. This strain is resistant to ccdB effects and can support the propagation of plasmids containing the ccdB gene. To maintain integrity for the vector, select for transformants in media containing 50-100 µg/mL ampicillin and 15-30 µg/mL chloramphenicol.
The BLOCK-iT Adenoviral RNAi Expression System includes the following features designed to enhance its biosafety:
- The entire E1 region is deleted in the pAd/BLOCK-iT-DEST expression vector. Expression of the E1 proteins is required for the expression of the other viral genes (e.g., late genes), and thus viral replication only occurs in cells that express E1 (Graham et al., 1977; Kozarsky and Wilson, 1993; Krougliak and Graham, 1995). This is where the Gateway Destination cassette is now located. The E3 region has also been deleted.
- Adenovirus produced from the pAd/BLOCK-iT-DEST expression vector is replication-incompetent in any mammalian cells that do not express the E1a and E1b proteins (Graham et al., 1977; Kozarsky and Wilson, 1993; Krougliak and Graham, 1995).
- 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 safety features discussed above, the adenovirus produced with this System 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 that express shRNA targeting human genes involved in controlling cell division (e.g., tumor suppressor genes) or when producing large-scale preparations of virus (See manual, pg. 11).
- 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) (http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm)
Adenovirus is not an actively lytic virus, meaning that mature viral particles accumulate in the cell over the course of two to three days. As virus accumulates, the producer cell rounds up and eventually bursts due to the sheer number of virus particles inside. Once this occurs, neighboring cells become infected and the three-day cycle begins again. The term “cytopathic effect”, or CPE, is used to describe this and is typically visible within approximately 7 days posttransfection in the form of “comet-shaped” plaques resulting from two rounds of infection, replication and cell burst.
After 7 days, CPE will expand and eventually take over the plate by approximately 10 days posttransfection.
10 days are required to produce virus from a transfected dish of cells (as just described).
Once an initial viral stock is produced, it can be amplified directly by infection of fresh 293A cells at a multiplicity of infection (MOI) of 3.
Please see the definitions below:
Infection: Applies to situations where viral replication occurs and infectious viral progeny are generated. Only cell lines that stably express E1 can be infected.
Transduction: Applies to situations where no viral replication occurs and no infectious viral progeny are generated. Mammalian cell lines that do not express E1 are transduced. In this case, you are using adenovirus as a vehicle to deliver shRNA.
Please see the steps below:
Clone the double-stranded DNA oligo encoding an shRNA or miR RNAi into one of the BLOCK-iT entry (shRNA) or expression (miR RNAi) vectors.
Transfer the RNAi cassette into the adenoviral (shRNA only) or lentiviral destination vector by Gateway recombination.
Transfect RNAi vectors into the viral producer cells to produce viral stocks, which can be used immediately or stored at -80 degrees C.
Harvest viral supernatants and determine the titer (amplify adenoviral stocks if desired).
Transduce lentiviral or adenoviral stocks to any cell type.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.