Custom Adeno-Associated Virus (AAV) Production Service

AAV Production Process

1. We begin by subcloning the gene of interest (GOI), shRNA or gRNA into a related pAAV cis-plasmid (optional).
2. Large-scale preparation of the pAAV cis-plasmid and complimentary plasmids using Qiagen Endo-free Mega Prep kits (optional).
3. Large-scale transfection of involved plasmids into 40x15cm plates of HEK293 cells.
4. Harvest the AAV production cells and purify the AAVs through a series of CsCl centrifiguations.
5. Determine the titer of the viral stock (in genome copy number per ml, or GC/ml) through quantitative real-time PCR.

A step-by-step diagram of our AAV custom production process.

What do I need to provide if I want the complete service from pAAV cloning?
For over-expression AAV construct, you need to provide us 5-10 ug plasmid DNA, the vector ma, and sequence for your plasmid.

For shRNA AAV service, you should provide the exact RNAi sequence to be cloned into recombinant AAV vector if no plasmid is available. If you already have the shRNA in a plasmid vector, we prefer to use your shRNA plasmid instead of just the shRNA sequence info.

For gRNA AAV, you can provide the gRNA sequence you want to use.  Please specify if your gRNA was designed for saCas9 or spCas9.

What do I need to provide if I already have my GOI/shRNA/sgRNA in pAAV vector?
In that case, we need 120-500ug of your pAAV plasmid along with the vector map/sequence for viral production. The exact amount of plasmid needed depends on production scale. We also recommend using endotoxin-free maxi or mega prep if possible.

If you only have small amount of AAV plasmid (or bacteria stab/glycerol stock), we can also start your AAV production from endotoxin-free plasmid prep of your AAV plasmid.

I don’t have any plasmid to send you, can you still make the AAV for me?
We can make AAV to over-express any genes (wildtype, mutant or synthetic) and to silence/knockout any genes from any species if you don’t have any starting material.

Our scientists are available to discuss your specific requirement and come up with the recommended AAV constructs for your specific needed. We can then produce the AAV based on the agreed specification.

How long does the entire process take?
The entire AAV production process takes ~4-5 weeks starting from AAV cloning.

If you perform the first subcloning and Qiagen Mega prep procedures for the cis AAV plasmid before sending it to us, our portion of the process will take ~2-3 weeks to complete.

Which serotype of AAV are available for custom production?
We can produce AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10 for wild type serotypes, and any synthetic/mutate serotypes like AAV-DJ, DJ8, DJ9, AAV6.2,  etc.

Is there a size limit for a gene to be delivered by AAV?
Yes, AAV has a packaging capacity of ~4.5Kb.

What options do I have for expression of cDNA, shRNA or gRNA?
You can choose from over 250 vector options for the expression of cDNA, shRNA or sgRNA. Please contact us for more information about these expression options to see which one best fits your research requirements.

What’s the expected yield and the storage solution?
For large scale production, expected yield is 1ml of >=1×10¹³ GC/ml of purified AAV stocks for most serotypes.  However, some genes are either toxic to cells or detrimental to AAV efficiency, and some serotypes (e.g, AAV 2, 3, 6) are low yield.  In these cases, the expected titer may be lower.  Our storage solution for all AAVs is PBS with 5% Glycerol, which is suitable for in vivo injection.

What type of QC do you have for each AAV stock?
The standard quality control includes qPCR for titration.

Do you offer different production scales for AAV?
Yes, We offer 3 different production scales for AAV – pilot scale, medium scale or large scale.