Adeno-associated virus (AAV) vectors have proven to be highly effective gene delivery tools for therapeutic research thanks to a variety of features, including minimal pathogenicity, sustained viral persistence, and the ability to target specific cells and tissue types with a variety of recombinant AAV serotypes (Büning 2019). In order to make good on this far-reaching potential, AAV vectors must be generated with high purity. However, many currently available schemas for their generation are serotype dependent, requiring separate method development and optimization for each vector. Our AAVpro ”All Serotypes” kits solve this issue through the use of a simple precipitation and filtration protocol (Figure 1) that can purify AAV particles with any capsid (i.e., from any serotype).
Serotype-independent, simple, and efficient purification method for Adeno-Associated Virus (AAV)
- Simple workflow: optimized protocol that eliminates the need for freeze-thaw cycles and ultracentrifugation
- High purity for reliable data: purified AAV particles are suitable for in vivo research
- Serotype agnostic: precipitation and filtration protocol that can purify AAV particles from any serotype
High purity for each AAV serotype
In order to test the purity of the system, AAV viral particles of each serotype (AAV1, AAV2, AAV3, AAV5, and AAV6) carrying the fluorescent protein ZsGreen1 were purified from producer cells using the AAVpro Purification Maxi Kit (All Serotypes). Viral titer was measured using real-time PCR analysis of the viral genomic sequence. This was followed by SDS-PAGE for each sample, and all serotypes displayed the AAV capsid proteins (VP1, VP2, and VP3) as the major bands present, which confirmed the purity of the AAV particles.
Infectivity of purified AAV particles
Each of the AAV samples purified above were then used to infect three different cell lines: CHO, RD, and HT1080. Three days later, flow cytometry was performed to assess infectivity. The resulting data showed that the AAV particles purified using this kit were able to infect the cell lines tested.
Infection of iPSC-derived cardiomyocytes
We further tested AAV viral particles purified with this kit by looking at their applications with iPSC-derived cardiomyocytes. Once again, viral particles carrying the fluorescent protein ZsGreen1 (serotypes 1, 2, 5, and 6) were purified from producer cells using the AAVpro Purification Kit Midi (All Serotypes), and titer of each purified vector was measured as described above. iPSC-derived cardiomyocytes were infected with either purified AAV1, 2, 5, or 6 vectors. The resulting ZsGreen1 fluorescence showed that the purified particles were indeed effective in this application, although AAV6, 2, and 1 appear more suitable for transducing iPSC-derived cardiomyocytes than AAV5.
The AAVpro Purification Kit (All Serotypes) can be used reliably for any AAV serotype with high purity and recovery in just four hours. The optimized protocol eliminates the need for freeze-thaw steps and ultracentrifugation, which are otherwise needed for extraction from cells and purification, respectively. We recommend browsing a collection of recent publications (listed below) to supplement your knowledge base for use of these "All Serotypes" kits for various in vivo research studies, including as direct injection into mouse brain.
Purity and infectivity of purified AAV particles
Viral particles from serotypes AAV1, AAV2, AAV3, AAV5, and AAV6, each carrying the fluorescent protein ZsGreen1, were purified from producer cells cultured in five T225 flasks according to the the AAVpro Purification Kit Maxi (All Serotypes) User Manual. Viral titer was measured using the AAVpro Titration Kit (for Real Time PCR) Ver.2 (Cat. # 6233). SDS-PAGE was performed on each sample, with 1 x 109 vector genomes loaded per lane.
The purified AAV particles carrying the fluorescent protein ZsGreen1 (above) were used to infect three different cell lines (CHO, RD, and HT1080) at 5,000 vector genomes per cell (serotypes 1, 2, 3, and 6) or 50,000 vector genomes per cell (serotype 5). Flow cytometry was performed three days later.
Infection of iPSC-derived cardiomyocytes
AAV1, 2, 5, and 6 vectors carrying the ZsGreen1 gene were purified from producer cells cultured in five T225 flasks with the AAVpro Purification Kit Midi (All Serotypes) kit, according to the user manual. Each purified vector was used for viral transduction of iPSC-derived cardiomyocytes at 500,000 vector genomes per cell. After 72 hr of infection, ZsGreen1 fluorescence was analyzed by microscopy, with an exposure time of 0.2 sec. AAV5 was additionally exposed at 0.5 sec. NC = negative control.
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