Helper-free expression system (CMV promoter)

AAVpro Helper Free Systems are kits for the preparation of AAV particles without the use of a helper virus. The AAV particles produced with these systems can be used to obtain transient expression of target genes from a strong, constitutive CMV promoter in mammalian cells or individual animals. When introducing genes to animals (in vivo transduction), purification with an AAVpro Purification Kit is required.

Preparation of AAV particles with the AAVpro Helper Free System

Each kit includes three plasmids encoding the factors necessary to prepare recombinant AAV particles by transfection of HEK293T cells. To prepare particles, cotransfect HEK293T cells with the three plasmids and 2–3 days later extract particles using the included AAV Extraction Solution, a reagent that allows 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.
• pRC (pRC1, pRC2-mi342**, pRC5, or pRC6): Each plasmid carries AAV Rep and Cap genes.
• pHelper: Plasmid that contains adenovirus E2A, E4, and VA, genes required for efficient AAV production.

*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 includes an expression cassette for has-miR-342, a human microRNA that increases AAV titer by approximately twofold as compared to ordinary pRC2 vectors that express only Rep and Cap.

Overview

• AAV particle preparation without using a helper virus
• Each AAVpro Helper Free System includes three plasmids encoding the factors necessary to prepare recombinant AAV particles and reagents for extracting AAV particles from producer cells
• AAV Extraction Solution is included with each AAVpro Helper Free System for fast, efficient extraction of viral particles; results in ~3X more harvested AAV particles than typical freeze-thaw methods
• The packaging plasmids (pRC and pHelper) are also sold separately (AAVpro Packaging Plasmid Sets) in large quantities (1 mg) for large-scale virus production

Applications