FAQ: Viral Expression
Q: How do I determine which viral vector to use for gene delivery?
A: There are several considerations when deciding on the best viral vector:
- Do I need transient or stable gene expression?
- Do I need to infect dividing or non-dividing cells?
- How important is potential immune response from my target cell?
- Will you be using your viral vector in vivo or in vitro?
You can find a quick comparison of viral vectors with respect to these and other factors in our Selection Guide.
Q: Measuring the titer of my virus is too complex and takes too long. Why should I bother?
A: Because viral gene delivery is a complex, multi-step process, occasionally you don’t get the gene expression you expect. Knowing your viral titer will help determine where the problem might be: during viral packaging, during purification, or during transduction of your target cell. While it’s true that most traditional methods of measuring viral titer are tedious and time-consuming, we offer convenient, user-friendly methods to determine viral titer for AAV, adenovirus, lentivirus and retrovirus.
Q: I have seen viral titer expressed in various formats: VP/mL, TU/mL, ifu/mL and pfu/mL? What is the difference?
A: There are two main types of viral titer:
- Physical titer is strictly a measurement of how much virus is present, and is usually expressed as the number of viral particles per mL (VP/mL).
- Functional titer, also known as infectious titer, is the measurement of how much virus actually infects a target cell. Functional titer may be expressed in the form of transduction units per mL (TU/mL), plaque-forming units per mL (pfu/mL), or infectious units per mL (ifu/mL), depending on the viral vector.
Functional titer is always a more accurate measurement because it measures how much virus actually gets into the target cell. However, functional titer usually takes much longer to determine and is sometimes not practical. In such cases physical titer may be measured, and functional titer may be calculated from physical titer. Functional titer will always be lower than physical titer, usually by a factor of 10 to 100-fold.
Q: What is the difference between purified and unpurified viral supernatant? Can filtering the supernatant with a low protein-binding filter be considered purification?
A: Purified viral supernatant means that it is free of serum protein and contaminants. Purification usually involves column chromatography or ultracentrifugation. Supernatant passed through a protein-binding filter simply removes cell debris and large insoluble particles. Such a supernatant would not be considered purified and would be particularly unsuitable for any in vivo use.
Q: Can your ViraBind™ AAV Purification Kit be used to purify various AAV serotypes such as AAV-2, AAV-5 and AAV-8?
A: Our ViraBind™ AAV Purification Kits are useful for AAV-2 and our own AAV-DJ. It will not work with AAV-DJ/8 or any other AAV serotypes.
Q: When collecting recombinant AAV, I discard the growth medium and later collect the viruses by disrupting the cells. With lentivirus, however, the cells are discarded and the growth medium is concentrated. Would it be helpful to concentrate the AAV growth medium via centrifugation as a possible source of additional recombinant AAV to add to the AAV in the cells?
A: Retrovirus and lentivirus mature differently than adenovirus and AAV. Retrovirus and lentivirus mature through a membrane budding mechanism, which is why you harvest them from conditional medium. Adenovirus and AAV package and mature all within the cell, so the cells are lysed via freeze/thaw cycles to release the virus. Very little adenovirus or AAV will be present in the conditional medium, so it is probably not worth the effort to harvest such a small amount. However, before discarding the medium it is a good practice to check the cells under a microscope to be sure they have not already been lysed.
Q: Can the filters provided in your ViraBind™ Adenovirus Purification Kit be re-used? How long can they be stored?
A: The syringe-type filters found in our ViraBind™ Adenovirus Purification Kit #VPK-100 are designed to be used twice. Please see the product manual for the protocol to regenerate the filter for the second use. The regenerated filter can be stored at 4ºC for one year. Be sure to pass some 1X Wash Buffer through the filter before the first loading of your viral supernatant. Each filter may only be used twice.
Q:Why are your ViraSafe™ Lentiviral Expression Systems safer than other systems?
A: ViraSafe™ Lentiviral Expression Systems introduce added safety measures in a couple of ways:
- ViraSafe™ Lentiviral Expression Systems are specifically engineered to minimize the chance of making replication-competent lentivirus (RCL). The ViraSafe™ systems use 3rd generation lentiviral packaging technology which provides for co-transfection of 4 separate plasmids, but sequence homology is reduced an additional 80-85%, making the chance of RCL even lower.
- In addition to the standard VSV-G pseudotyped virus, which can infect virtually any cell regardless of species, you can now make an ecotropic lentivirus by choosing a ViraSafe™ Ecotropic Expression or Packaging System. Viruses made with an ecotropic system are packaged with a different envelope protein that will only readily infect mouse and rat cells.
Q: Can I use one of your Platinum Retroviral Packaging Cell Lines with a lentiviral expression vector containing my gene?
A: Even though lentivirus is a sub-class of retrovirus, the structural genes of HIV and MMLV are different enough that they are not interchangeable and therefore will not produce a viable viral particle.
