Adeno-associated virus (AAV) is a popular choice for delivering genes to cells in vivo. Several features of the virus, including minimal pathogenicity, sustained viral persistence, and broad cell-type infectivity, make AAV-mediated gene delivery an effective strategy for creating disease models and studying gene function in animal models.

When preparing AAV particles for in vivo transduction, purity and titer are critical for achieving high transduction efficiency. Purification of the virus is essential when it is to be used in vivo, as crude viral lysate can be toxic to the target tissue/cells. In addition, having an accurate estimation of viral titer ensures that sufficient virus is delivered and helps maintain consistency between experiments. In this example, AAV2 particles for in vivo transduction of brain cells were generated using the AAVpro series, a set of products for generating high titers of transduction-ready, recombinant AAV particles. Using this system, AAV2 particles were successfully used for in vivo delivery of a bright fluorescent marker to cells in a very specific brain region in mouse.

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Strong green fluorescence was observed throughout the striatum (left panel). Also, ZsGreen1 expression was detected in the neurons of the globus pallidus (right panel).

ZsGreen1 expression in a coronal brain section. The left panel shows the entire brain section (scale bar, 1 mm); strong fluorescence in the striatum is noted. The right panel is an enlargement of a portion of the left panel (scale bar, 30 µm), showing expression in the globus pallidus. Data credit: Prof. Muramatsu Shinichi (Jichi Medical School).

The purity and titer of AAV particles is important for achieving high transduction efficiency into individual animals. AAV2 particles obtained using the AAVpro system could be used for efficient in vivo transduction into mouse brain.

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AAV2 particles were prepared using the AAVpro Helper Free System (AAV2) and pAAV-ZsGreen1 according to the recommended protocol. The Helper Free System provides a unique method for the preparation of high-titer AAV particles that does not require the use of a helper virus. This kit includes plasmids encoding the factors necessary to prepare recombinant AAV particles by transfection of HEK 293T cells. pAAV-ZsGreen1 is an AAV vector that can be used to deliver an exceptionally bright green fluorescent protein to target cells.

To generate the AAV2-ZsGreen1 particles, 293T cells were seeded the day before transfection. pRC2-mi342 and pHelper plasmids from the AAVpro Helper Free System (AAV2) kit, and pAAV-ZsGreen1 were introduced using the calcium phosphate transfection method as described in the AAVpro Helper Free System (AAV2) user manual. Forty-eight hours after transfection, the cells were detached using 0.5 M EDTA (pH 8.0).

AAV2 particles were purified using the AAVpro Purification Kit (AAV2), a simple and fast purification method that does not use ultracentrifugation. Briefly, the packaging cells were collected by centrifugation and viral particles were extracted using the AAV Extraction Solutions. Then, the extract solution was treated with Cryonase Cold-active Nuclease and the solution was added to the pre-packed purification column. After washing, particles were eluted from the column. The eluted solution was desalted and concentrated to obtain purified AAV2-ZsGreen1 viral particles. Following purification, the AAVpro Titration Kit (for Real Time PCR) was used to measure the AAV2-ZsGreen1 virus genomic titer. 2.4 × 10¹² vector genomes (vg) of purified AAV2-ZsGreen1 was obtained.

The purified AAV2-ZsGreen1 particles were diluted to 1.86 × 10¹² vg/ml in PBS, and 5 µl was administered to the brains of mice at points to the left and right of the striatum using a stereotactic surgical technique. After 27 days, anesthetized mice were perfused with fixative (a solution containing 4% paraformaldehyde), and brains were removed. After freezing, coronal thin sections were prepared. Slices were imaged by fluorescence microscopy.