Q: What is a bicistronic expression system?
A: A bicistronic vector contains an IRES element that controls the translation of a downstream gene, such as GFP. This means that translation of the GFP mRNA is independent of translation of the gene of interest that is cloned into the MCS, which is typically cap-dependent translation. This allows for separate expression of your protein and GFP as well as better stable selection if using an antibiotic resistance gene. The downside is that IRES expression is usually weaker.
Q: Which AAV serotype should I use?
A: The best serotype for your specific target cells can be determined through a literature search, or you can use this table which shows relative infectivity levels for different cell types. We offer native AAV serotypes 1-6 as well as the AAV-DJ and AAV-DJ/8 systems licensed from Stanford University. Research shows that AAV-DJ/8 has good in vivo infectivity rates in brain and heart tissues, while AAV-DJ has been shown to be very good for in vitro transduction. Click here for a link to the original paper.
Q: What vectors are required for packaging AAV?
A: AAV packaging using the AAV Helper Free Systems requires an expression vector, a pHelper vector, and a serotype-specific rep-cap vector.
Q: Do you have a recommended bacterial strain for amplifying AAV packaging plasmids?
A: Our AAV vectors can be amplified using E.coli competent cells. For amplification of the expression vector, we recommend using Invitrogen Stbl3 competent cells which will minimize recombination that can occur through the ITRs and result in a plasmid of reduced size. If Stbl3 cells are not used, a restriction digest can be performed to confirm the plasmid has maintained the correct size. Recombination is not a concern with the packaging plasmids, which do not contain ITRs. These plasmids can be amplified using standard protocols.
Q: How much total DNA should be used for transfection and at what ratio?
A: We recommend following the manufacturer’s protocol for the transfection reagent you will be using, which will have guidelines for the amount of DNA to use. A 1:1:1 vector ratio should be used.
Q: Do you have recommendations for the calcium phosphate transfection protocol?
A: Calcium phosphate may be used if you want to minimize the cost when scaling up AAV production. Calcium phosphate transfections can be difficult because the pH is very critical and a difference in pH of 0.05 can affect the transfection. We recommend preparing the HBS buffer with a range of pH values in 0.05 increments and testing in parallel.
Q: Can HEK293 cells be used to package virus?
A: Yes, HEK293 cells can be used to package AAV-DJ. All 293 cells are E1 transformed HEK cells, therefore any 293 cells will be able to package AAV when cotransfected with an expression vector, a pHelper vector, and a rep-cap vector. Most 293 cells don’t adhere well and detach easily when they become confluent, which can result in a low AAV yield. If this is a problem, our 293AAV cell line (AAV-100, http://www.cellbiolabs.com/aav-host-cell-line) can be used, which was derived from parental HEK293 cells and were specifically selected to have flattened morphology and firm attachment to improve viral applications.
Q: Can the AAV expression vector be used for transient transfection?
A: The AAV expression vector can be transfected into cells without the packaging vectors to measure gene expression. This will be a transient transfection and therefore will not be stable, but will allow you to confirm that your construct is working as intended.
Q: When should the cells be harvested after transfection?
A: We recommend harvesting between 48-72 hours because most virus is made during the first 48 hours and very little additional virus is made after 72 hours.
Q: Why do I not see any morphological changes to the cells 48 hours after transfection?
A: It is normal not to see any morphological changes to the cells upon viral production. Transfection with a GFP expressing plasmid can confirm transfection efficiency, but there will not be noticeable cytopathic effects to the cells.
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: What is a typical expected titer for AAV?
A: With optimized transfection conditions, a typical titer from one 15cm dish is 1-2x1011 genome copies (GC) or 1011 to 1012 GC/mL, with each cell producing 2x104 to 1x105 viral particles. There are many variables that can influence this value, such as the size of the insert, the packaging cells, and the transfection efficiency.
Q: How can I improve the titer of my AAV?
A: Here are some suggestions to improve the titer:
1. We recommend using the GFP control virus to monitor transfection efficiency and to confirm that virus is being produced. The transfection conditions may need to be optimized until at least 80% transfection efficiency is achieved after two days.
2. It is important to use 293 cells that are healthy and at a low passage number.
3. Recombination can happen after transformation through the ITRs, resulting in a plasmid of reduced size. We recommend using Invitrogen Stbl3 competent cells for plasmid amplification, which will minimize recombination. If Stbl3 cells were not used for plasmid amplification, you should confirm that the plasmids have maintained the correct size by running a restriction digest.