FAQs

You can read about the primary differences between the two viral vectors here.

The recombinant adenoviruses we produce are replication deficient due to deletions in the E1 and E3 regions. According to references issued by the NIH Office of Biosafety, recombinant human adenoviruses are classified as biosafety level II for agents considered ordinary potential harm.  You need a BL-2 level facility to work with them. It should be noted that cell culture facilities in most institutes are certified as BL-2 level.

Wild type, replication competent adenoviruses provoke cold symptoms and strong immune responses in healthy individuals but generally do not cause serious illness.

For more information on biosafety levels, please read the NIH Biosafety Guidelines.

The adenovirus has a very broad host range; it can infect human and other mammalian cell lines or primary cells, including replicative as well as non-replicative cells. In fact, there are very few cell lines that cannot be infected. Some lymphoid cell lines may be more resistant to adenoviral infection, and may need higher viral quantities to achieve sufficient infection levels.

For your convenience, we offer some marker adenoviruses, such as Ad-CMV-ß-Gal or Ad-CMV-GFP to allow you to conduct pilot testing in your systems.

The appropriate amount of active/infectious viruses used for infecting cells is very important for the outcome of your experiments. If not enough virus is used, you will not reach 100% infection. If too much is used, it will result in cytotoxicity or other undesired effects. You should use the minimum viral concentration that will result in 100% gene delivery. This optimal concentration differs dramatically between cell types. To determine this optimal viral concentration, you could conduct pilot testing in your system by using marker adenoviruses, such as Ad-CMV-ß-gal.

We have tested numerous cell types by exposing cells to virus-containing media for 6-8 hours or overnight. For most cell types, viral concentrations of 2 x 10⁵ – 1 x 10⁶ IFU/PFU (infectious unit)/ml of media results in 100% infection without visible side effects. However, we recommend you test your cell system first by using marker viruses.

For your reference, we recommend the following amount virus-containing media for infection:

• 10-cm plate: 8-10 ml per plate
• 6-well plate: 1.0 ml per well
• 12-well plate: 0.5 ml per well
• 24-well plate: 0.2 ml per well

This roughly reflects the surface area of each well or plate.

Viral Particles (VP) represent the total number of both live and dead viral particles. Due to variations in virus preparations, the ratio of live/dead varies significantly. Therefore, VP does not reflect the amount of active virus in the preparation.

Plaque Formation Unit (PFU) represents the number of infectious or live viruses. It reflects the amount of working viruses in the preparation.

Infectious Unit (IFU) is equivalent to PFU.

For most virus preparations , the VP/PFU ratio is 20:1 to 50:1.

Using Viral Particles (VP) as measurement will result in significant variations in the amount of actual live viruses present, whereas using IFU or PFU as the viral unit will give more consistent outcomes.

There are 3 commonly used protocols for determining adenovirus titer: (1) OD260 Assay, (2) Plaque Formation Assay, and (3) End-point Dilution Assay.

OD260 assay measures the concentration of viral DNA and protein. It does not distinguish between intact, infectious viruses and damaged, non-infectious viruses. It is a physical assay measuring the concentration of total viruses, both live and dead. Based on OD260, the concentration of viral particles (VP) could be obtained. To measure OD260, the virus stock has to be purified first.

On the other hand, Plaque Formation Assay measures the concentration of infectious viruses, and therefore is a biological assay. Essentially, a mono-layer of HEK293 cells are infected with a series of virus dilutions. Viruses will propagate in the infected cells, and eventually cause cytotoxicity effects before being released. The released viruses will infect neighboring cells, and the whole process will be repeated, eventually leading to the formation of holes (or plaques) on the cell monolayer. In order to prevent the diffusion of viruses and plaque formation, top agarose is layered on top of cells after the initial infection.

The biological principle for End-Point Dilution Assay is similar to the Plaque Formation Assay, although the procedure and measurement is different, and the formula for calculating the virus titer is more complicated.

Although both the Plaque Formation Assay and End-Point Dilution Assay gives the titer of infectious or working viruses, they are scored by the human eye and subject to human and procedural variations. For the same virus stock, it is not uncommon that two different people will get significantly different titer readings.

No. If the viruses will be used in in vitro cell cultures, double CsCl purification is not required. For in vivo studies (i.e. animal studies), purification is essential in order to remove defective particles, cell debris, and small amounts of media components, as these contaminants induce significant immune responses. In addition, CsCl purification will concentrate the virus to a level suitable for in vivo injections.

For long-term storage, the virus should be kept at -80°C, especially after CsCl or chromatography purification. At -80°C, the virus should be stable for 6 months to a year (and in some cases, up to 2 years).

If the virus is in DMEM supplemented with serum or BSA and stored at -80°C, it will remain stable for longer periods of time and through several freeze-thaw cycles.

On the other hand, if the virus CsCl-purified and kept in PBS or Tris solution (20mM Tris PH8.0, 200 NaCl, 2-3% glycerol or sucrose), repeated freeze-thaw cycles should be avoided, as it will cause a significant decrease in titer.

The cloning capacity for the transgene, by using (DE1/E3) adenovirus type 5, is about 8 Kb in length.

The most commonly used adenovirus for gene delivery is human adenovirus serotype 5 (DE1/E3), which we produce.

One concern when working with adenoviral vectors is the rare occurrence of replication-competent adenoviruses (RCAs) in a population of replication-deficient viruses. RCAs can emerge from a rare double crossover through overlapping sequences present in the recombinant adenovirus and the genome of HEK293 cells. This results in replacement of the transgene by the E1 region. Once this happens, the adenovirus can replicate without a complementing cell line. To detect RCA, non-complementary cells (such as A549 cells) are incubated with the viral stocks (1E4-1E6 PFU) and monitored for cytopathic effects (CPE) and/or plaque formation. According to NIH guidelines, less than 1 plaque in about 1E4 viruses is considered safe to use. To avoid the occurrence of RCAs, viruses should be produced and amplified in low passage packaging cells.

By Phone: 1-484-325-5100, option 1
By Fax: 1-215-525-1112
By Email: order@vectorbiolabs.com

If you have a Purchase Order, you can just send us a copy of it via email or fax.  If you wish to pay by Credit Card, please fill out our Credit Card Order Form.  Please note, that a 3% transaction fee will be applied to orders placed via credit card.

Orders are normally shipped out the same day if the product is in stock and we receive the order before 1PM Eastern Time.  If the product is not immediately available, you will receive email updates as production progresses.

The buyer is responsible for any duties and tax fees required by your country for importing consumer goods. We do not collect this beforehand, and cannot give you an estimate of the cost – charges vary country to country.

Your order will be shipped in dry ice package. We use FedEx “Standard Overnight” shipping for US orders, and “International Priority” option for non-US orders. If you want to use a different shipping option, please contact us and an additional charge may be applied to your order.

Yes, if you provide us with your FedEx account at the time of ordering, we can ship using your FedEx. In this case, we will not charge you the shipping cost, only a dry ice packaging cost.

Yes. But orders have to be placed with us directly since we do not have local distributors outside US at this time.

Yes. Please contact us for conditions and limitations for distributors.

A surcharge of $35 USD per product will be applied to each catalog product for international orders. For example, if the listed price for a product is $475 USD, the international price would be $510 USD after the surcharge. International pricing applies to all orders placed outside US/Canada.

Just email us your name, name of your institution, contact phone number, and the catalog number and quantity for each product you want to order, and we will email you an official quote in PDF file.

If you are a US customer outside the state of Pennsylvania, we don’t charge sale tax for your state. However you may still be responsible for the sale/use tax for your state, which you should pay directly to your state.

If you are a customer in state of PA, please send us a copy of your tax exempt certificate, so we will not charge the sale tax for your order.

For international customers, we don’t charge VAT for your orders.

We are not responsible for determining what import permits are needed, since every country has their own rules and regulations. Please contact the appropriate person at your institute or customs office to determine what is required before placing an order.

Vector Biolabs
Attn: Tech Services
293 Great Valley Parkway
Malvern, PA 19355, USA
ph: 1-484-325-5100

We recommend using an express carrier such as UPS or FedEx instead of the postal service. If possible, we strongly suggest using overnight service (or the fastest available if shipping internationally) to minimize transit time.

We require at least 10ug if we need to start from cloning.

For pAd plasmids, a minimum of 20ug is required for adenovirus packaging.

For pAAV plsamids, 120 – 500ug pAAV plasmid (endotoxin-free prep preferred) is required for AAV viral packaging depending on production scale, unless you would like us to do the plasmid preparation for you.  In that case, we only need a small amount (1-5ug) of plasmid DNA or bacteria glycerol stocks.  Here are the amount of DNA needed for viral packaging for each production scale:
Pilot Scale:          120ug
Medium Scale:    250ug
Large Scale:        500ug

The concentration of your plasmid DNA should be >=0.5ug/ul.

Yes, if your plasmid is stored in a TE buffer, you can ship them to us at room temperature. Otherwise, we recommend using a cold package (blue or dry ice) for shipment, in order to reduce the likelihood of DNA degradation.

We work with hundreds of labs for thousands of viral constructions and receive plasmids every day. To ensure use of the correct plasmid for your construct, we require a submission form for each plasmid sent to us.

For non-viral plasmids, we need a vector map of the plasmid and sequence of the vector backbone (if available).  We also need the sequence of the gene of interest (GOI), so we can design the best cloning strategy for viral construction.  This information should be emailed to cloning@vectorbiolabs.com.

For pAAV cis-plasmid, please also email us map/sequence for your plasmid. We need to pick the right qPCR primers for titration the AAV stock after production.

Your project may be delayed if insufficient information is provided for your plasmid.

If you receive the plasmid from an outside source, we recommend doing some quick sequencing to ensure it’s the correct plasmid with the correct insert.