Frequently Asked Questions

The answer to this question typically depends on whether you are planning to do a direct injection or you need an engineered capsid that crosses the blood-brain barrier OR an engineered capsid happens to work better for injecting your target tissue. For example, if you want to target a small region on the brain, then it is best to use a natural AAV serotype such as AAV8 or AAV9 and directly inject into that site. If you would like a broader distribution and want to take advantage of capsid engineered AAVs, then we would recommend PHP.eB or CAPB10 for brain-wide transduction. CAPB10 has the advantage of liver de-targeting (Goersten et al., 2002). Most of what we know about BBB crossing capsids comes from studies in C57BL/6 mice. Other BBB capsids, such as PHP.eC, work well in non-C57 strain backgrounds. 

Please reach out to Andrew Steele with questions regarding serotypes and capsids. So far, for natural serotypes, we offer AAV2, AAV8, and AAV9. We have a variety of Gradinaru lab engineered capsids, including PHP.AX, PHP.S, PHP.eB, PHP.eC, X1.1, MaCPNS1, MaCPNS2, and CAPB10. Some of these capsids are excellent for targeting peripheral nervous system while others are mainly useful to CNS targeting. 

Yes: almost everything we produce is a custom AAV preparation tailored to the needs of a specific project.

For AAV preps, we use a standard triple-transfection approach. This involves co-transfecting HEK cells with three plasmids: a helper plasmid, an AAV rep/cap plasmid that determines the serotype (for us this is almost always a Gradinaru lab AAV9 derivative), and the pAAV cargo plasmid containing the expression cassette to be packaged. The ArmVC already has the helper plasmid and commonly used rep/cap plasmids readily available. In most cases, the only plasmid we need from the user is the pAAV cargo plasmid.

Users can provide their own pAAV plasmid, ask us to order an available construct from Addgene, or work with us to generate a new custom construct. For custom builds, we commonly coordinate plasmid design and synthesis through VectorBuilder. Once the cargo plasmid is available and sequence-confirmed, we can move forward with AAV production.

If you are trying something new, it is best to start with a smaller scale preparation. Our minimum scale prep is 5-plates (15 cm dishes). With an average yield, this should give at around 2.5-5E12 total vector genomes (vg). If you are injecting a standard dose in mice of 3E11 vg, that would give enough to test 8 mice on the low end and 16 mice on the high end. 

For a 10-plate preparation, we expect to obtain between 5E12-1E13 total vg. Volumes are not important here--our viruses are concentrated and we often dilute them down to 1E10 vg/ul (or 1E10 vg/ml). If you need the virus more concentrated than that, please let us know. For example, a preparation at 1E10 vg/ul would still need to be diluted before doing a systemic injection in mice. We typically inject in a total volume of 50ul. 

We can do larger preparations, up to 80-plates per run. This scale if for doing 100's of mice or larger animals such as non-human primates. A good 80-plate preparation can give up to 8E12 total vg. A typicaly dose for a marmoset (400 grams) is 2.8E13 vg. 

For constructs that we have worked with before--please see the order tab for cargos/capsids that are in our catalog--we can guarantee >5E12 total vg from a 10-plate scale preparation unless we have found that this particular cargo does not produce well.

For cargos that we have not worked with before, we cannot commit to a particular yield. There have been a few instances where the cargo expression has caused toxicity to HEK cells or the cargo itself may have interfered with AAV production/assembly. Our goal is always to help you get your experiment done, so in cases when cargos were problematic, we assisted with Cre-dependent expression strategies to get the AAVs produced. 

We work with lots of first time AAV users and pride ourselves on going above and beyond to advise new users and help them be successful. We have extensive experience ourselves as intracranial and systemic AAV users and we have a particular interest in building recombinase dependent cargos in AAVs, including Cre, Flp, Dre, and VCre recombinases.

No. AAV preparations at the scale and purity needed for research experiments are produced by trained ArmVC personnel, not undergraduate trainees. We have found that custom AAV preparations intended for experimental use require a level of technical consistency, documentation, and quality control that should not be assigned to undergraduate trainees.

Undergraduate trainees have contributed to some of our catalog preparations as part of their training. However, these preparations are quality controlled before release, including validation by in vivo injection when appropriate. QC images and related information are available through our ordering platform so users can review the validation data associated with catalog preparations.