Are the BLOCK-iT™ miR RNAi Expression Kits compatible with adenoviral expression systems?
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 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
How does the adenoviral system work? How do I make an adenovirus expressing my gene of interest?
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.
Answer Id: 4119
Does the ViraPower™ Adenoviral Expression System use an adeno-associated virus?
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.
Answer Id: 4120
Is long-term expression of my recombinant protein possible using adenovirus?
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 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.
Answer Id: 4122