The AAVpro Helper Free System (AAV2) is a kit for the preparation of serotype 2 AAV (AAV2) particles without the use of a helper virus. The AAV2 particles produced with this system can be used to obtain transient expression of the target gene in mammalian cells or individual animals. When introducing genes to animals (in vivo transduction), purification with the AAVpro Purification Kit (AAV2) is required.
Preparation of AAV2 Particles with the AAV Helper Free System
The AAV Helper Free System is a unique system for the preparation of high titer AAV particles without the use of a helper virus. This kit includes three plasmids encoding the factors necessary to prepare recombinant AAV particles by transfection of HEK293T cells. To prepare particles, co-transfect HEK293T cells with the three plasmids, and 2-3 days later extract particles using the included AAV Extraction Solution, a reagent that allow simple and efficient AAV particle isolation while minimizing protein and nucleic acid contamination.
- pAAV-CMV: Plasmid for cloning the gene of interest (GOI)*. Includes a CMV promoter for gene expression and two ITRs.
* The size of the target gene cloned into the pAAV-CMV vector should be <2.5 kb as there is a limit to the size of DNA that can be encapsulated in AAV particles.
- pRC2-mi342: Plasmid that carries the Rep and Cap genes of AAV2 and an expression cassette for has-miR-342*
* hsa-miR-342 is a human microRNA that increases AAV2 titer in this vector preparation system. It increases titer by approximately 2-fold as compared to ordinary pRC2 vectors that express only Rep and Cap.
- pHelper: Plasmid that contains adenovirus E2A, E4, and VA, genes required for efficient AAV production.
Note: The packaging plasmids (pRC2-mi342 and pHelper) are also sold separately in very large 1 mg quantities (AAVpro Packaging Plasmid, Cat. No. 6234), which are convenient for large-scale production for in vivo studies.