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AAV Production Service
No DNA using our proprietary AAV plasmids is delivered.
Additional fee for DNA scale up is charged for BS090B-D.
AAV Purification: iodixanol centrifugation. (optional) Column chromatography.
Quality control: titering by qPCR. (optional) Endotoxin measurement and reduction, SDS PAGE, TCID50 assay.
Our complete custom Adeno-associated Virus (AAV) production services include: 1) AAV plasmid vector construction; 2) helper-free AAV packaging; 3) AAV purification by iodixanol gradient ultracentrifugation or column chromatography; 4) AAV titering by qPCR. Our professional AAV production team members have successfully produced high quality AAV with high titer from small to large scales for worldwide clients in more than a decade. We have always optimized our AAV production systems, including AAV plasmid construction and low endotoxin plasmid preparation, host cells for AAV packaging, cell culture medium and AAV packaging process, small and large scale AAV purification process.
The recombinant AAV (rAAV or AAV below) is currently among the most frequently used viral vectors for gene therapy, cell therapy (knock-out and knock-in cell line generation with CRISPR genome editing and Chimeric Antigen Receptor T (CAR T)-cell engineering with lentivirus or alone), and academia research studying gene expression and silencing in mammalian systems by gene knock-in and knock-out. AAV is ideal to be used as the vehicle for gene transfer and the tool for genome editing because:
- AAV is simple and easily manipulated;
- AAV can deliver genes to both dividing and non-dividing cells, and a wide variety of tissue types from muscle to brain;
- AAV is lack of pathogenicity and elicits very low immunogenicity only;
- AAV allows expression of proteins in host cells for long period of time.
BS090A. Molecular construction of an AAV vector and maxi-scale AAV virus production.
Gene-of-interest (GOI) sequences are subcloned into our proprietary AAV production plasmids or plasmid vectors that clients provide.
* Multiple serotypes (AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9).
* Wide choice of AAV cis vectors (promoterless, CMV, PGK, UBC or U6 promoters with or without reporter genes like GFP, mCherry, strawberry, Luc, LacZ, etc).
* From small crude scale to large purified scale. For example, midi-scale AAV transfection of HEK 293 cells in 20 x 150 mm plates.
* AAV purification via iodixanol centrifugation or (optional with additional fee) column chromatography.
* AAV titration (measuring genome copies) by qPCR. (optional with additional fee) Endotoxin measurement and reduction, SDS PAGE, and TCID50 assay. If endotoxin reduction is needed, please notify us before plasmid preparation for AAV virus production is initiated. If a purified recombinant AAV has high titer but shows lower transduction efficiency than one expects, it is highly possible that there is something wrong in the process of the qPCR method used for AAV titering. Many AAV production service suppliers do not realize the problem in overestimated AAV titration results.
2-3 weeks. Longer if quality control tests in addition to qPCR titering are performed.
2.0 ml purified viral stocks (1 x 10^12-13 GC/ml). Final viral yield may depend on the nature of transgene. If needed, the purified AAV can be concentrated to 1 to 5 x 10^13 GC/ml.
Cesium chloride (CsCl) density gradient ultracentrifugation is widely used for purification of AAV vectors in laboratory scale. AAV vectors purified by CsCl gradient centrifugation have been evaluated in many preclinical and clinical studies. CsCl is toxic to cells and must be removed prior to HPLC. Iodixanol is non-toxic to cells and rarely needs removing prior to further processing, except for electron microscopy studies. Compared to the fact that CsCl gradients lead to major reductions in viral infectivity, viruses from iodixanol gradients show a higher percentage recovery of infectivity and much lower average particle/infectivity ratios.
To avoid disadvantages associated with AAV purification using the ultracentrifugation techniques, chromatography-based systems have been developed with high yield, high purity, and high infectivity. Though it is more expensive to use chromatography to purify small scale of AAV, chromatography-based AAV purification methods are good for large scale production. Syd Labs provides the process development service for large scale manufacturing of recombinant adeno-associated viral vectors for clinical trials.
References of AAV production protocols, AAV purification protocols, and AAV titration protocols:
A protocol for AAV vector production and purification
A method used to propagate and purify AAV vectors for experiments both in vitro and in vivo.
Production and Titering of Recombinant Adeno-associated Viral Vectors
A method for production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2.
Inexpensive, serotype-independent protocol for native and bioengineered recombinant adeno-associated virus purification
A standard AAV virus purification protocol independent of toxic compounds, supernatant volume and capsid moiety, and across native serotype and bioengineered mosaic capsids.
The procedure of AAV production sounds very straightforward. However, many factors affect the efficiency and success rate. Experienced scientists at Syd Labs provide comprehensive AAV virus production services if the project is difficult for you.
We index "References of AAV production protocols, AAV purification protocols, and AAV titration protocols" for your information only. It does not necessarily mean that we agree with all of them. You rather than Syd Labs takes full responsibility for using any information described here.)
Syd Labs also provides the following virus production services (virus vector construction, virus packaging, virus amplification, virus purification, and virus titering):
Retrovirus Production Services
Lentivirus Production Services
Adenovirus Production Services
Adenovirus Production Services for Toxic Genes
Adenovirus Production Services for Fiber-modified Ad5-RGD and Ad5/35