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FAQs for recombinant adenoviruses

  • What is the required biosafety level for using recombinant adenovirus?
  • How can I find out whether adenovirus will work well in my cell models
  • What's the optimal concentration of viruses that I should use for infection?
  • How much media should I use during infection?
  • What are the differences between viral particle (VP), plaque formation unit (PFU) and infectious unit (IFU)? Which one of these better reflects the amount of active virus used?
  • How are virus titers determined?
  • For in vitro use (cell culture studies), is CsCl- or chromatography purification required?
  • What are the recommended storage conditions of recombinant Adenoviruses?
  • What is the capacity of cloning into the Adenovirus as an expression system?
  • There are many serotypes of adenoviruses, which one is the most commonly used for gene delivery?
  • What are RCAs?
  • There are several viral gene delivery systems, including adenovirus, retrovirus and lentivirus. Which one should I use for my experiments?

    Q. What is the required biosafety level for using recombinant adenovirus?

    The recombinant adenoviruses we made are replication deficient due to deletions in the E1 and E3 regions. According to references issued by the NIH Office of Biosafety, recombinant human adenovirus has been classified in biosafety level II for agents considered of ordinary potential harm, and you need BL-2 level facility to work with it. 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 and generally do not cause serious illness. For more information on biosafety levels please visit http://bmbl.od.nih.gov

    Q. How can I find out whether adenovirus will work well in my cell models?

    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. There are in fact very few cell lines that cannot be infected. Some lymphoid cell lines may be more resistant to Adenovirus infection, and so may need high quantities of viruses 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.

    Q. What's the optimal concentration of viruses that I should use for infection?

    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, it will not give 100% of infection. If too much virus is used, it will cause cytotoxicity or other undesired effects. You should use the minimal virus concentration that will give 100% gene delivery. This optimal concentration differs dramatically between cell types. To determine this optimal concentration of virus, 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 gives 100% of infection wthout visible side effects. However, we recommend you test your cell system by using marker viruses.

    Q. How much media should I use during infection?

    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 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.

    Q. What are the differences between viral particle (VP), plaque formation unit (PFU) and infectious unit (IFU)? Which one of these better reflects the amount of active virus used?

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

    PFU (plaque formation unit) represents the number of infectious or live viruses. It reflects the amount of working viruses in the preparation.

    IFU (infectious unit) is equivalent to PFU.

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

    Using VP (viral particles) as the unit will result in significant variations in the amount of actual live viruses used, and using IFU or PFU as the viral unit will give more consistent outcomes.

    Q. How are virus titers determined?

    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, live and dead. Based on OD260, the concentration of viral particles (VP) could be obtained. To measure the OD260, the virus stock has to be purified first.

    On the other hand, plaque formation assay measures the concentration of infectious viruses, and therefore it is a biological assay. Basically, a mono-layer of HEK293 cells are infected with a series of virus dilutions. Viruses will propagate in infected cells, and eventually cause cytotoxicity effects in that cell, and get 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 to other areas of the cells and start a new plaque formation process, a layer of top agarose is laid 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 a bit 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 subjected to human and procedure variations. For the same virus stock, it is quite common that different people will get significantly different titer readings.

    Q. For in vitro use (cell culture studies), is CsCl- or chromatography purification required?

    No. If 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, since these contaminants induce significant immune responses. In addition, CsCl purification will concentrate the virus to a level suitable for in vivo injections.

    Q. What are the recommended storage conditions of recombinant Adenoviruses?

    For long-term storage, the virus should be kept at -80°C, especially after CsCl or chromatography purification. At -80°C, the virus could be stable for 6 months to a year and in some cases even 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-and-thaw should be avoided, since it will cause significant decrease of titer.

    Q. What is the capacity of cloning into the Adenovirus as an expression system?

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

    Q. There are many serotypes of adenoviruses, which one is the most commonly used for gene delivery?

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

    Q. What are RCAs?

    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 as a result of the rare double crossover through overlapping sequences present in the recombinant adenovirus and the genome of HEK293 cells. This event results in the replacement of the transgene by E1 region. Once this happens, the adenovirus could replicate, without the need of 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 guideline, <1 plaque in about 1E4 viruses is considered safe to use. To avoid the occurrence of RCA, viruses should be produced and amplified in low passage packaging cells.

    Q. There are several viral gene delivery systems, including adenovirus, retrovirus and lentivirus. Which one should I use for my experiments?


    Adenovirus: 100% gene delivery efficiency in most cell types including dividing and non-dividing or primary cells. There is no integration with the host system. However, protocols involved in developing recombinant adenovirus are very complicated.

    Retrovirus: gives to <30% efficiency in most cell types, and requires active cell division. In addition, there is a significant risk of integration into the host genome, leading to mutation of genes or activation of onco-genes in the host system, which is a concern for scientists. Unlike adenovirus, recombinant retrovirus is very easy to develop.

    Lentivirus: gives to <30% efficiency, in both dividing and non-dividing cells. Like retrovirus, there is a significant risk of integration into the host genome, leading to mutation of genes or activation of oncogenes in the host system. As with retrovirus, recombinant lentivirus is very easy to develop.

    If you are interested in delivering your specific gene or siRNA into all cells, adenovirus system is the best choice. However, if you are interested in single-cell level studies, retroviral system, lentiviral system or even direct transfection of expression plasmid DNA may sometimes serve your need.

     

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