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Tech Note

Gene transfer into the mouse striatum via recombinant AAV2 particles generated using the AAVpro series

Data kindly provided by Prof. Muramatsu Shinichi, Division of Neurology, Jichi Medical University.

Introduction Results Conclusions Methods

Introduction  

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.

Learn more about the AAVpro workflow »

Results  

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 panel A (scale bar, 30 µm), showing expression in the globus pallidus. Data credit: Prof. Muramatsu Shinichi (Jichi Medical School).

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).

Conclusions  

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.

Learn more about AAVpro products »

Methods  

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.

Related Products

Cat. # Product Size License Quantity Details
6230 AAVpro® Helper Free System (AAV2) 20 ug Each USD $894.00

License Statement

ID Number  
M84 This product is the subject of the claims of US patent No. 10072250 and its foreign counterparts.
M83 This product is the subject of the claims of US patent No. 9422576 and its foreign counterparts.

Kit for the preparation of AAV particles with the AAV2 serotype, without the use of a helper virus. The AAV particles produced with this system can be used to obtain transient expression of target genes in mammalian cells or individual animals. 

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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pRC2-mi342 vector map

pRC2-mi342 vector map
pRC2-mi342 vector map.

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pHelper vector map

pHelper vector map
pHelper vector map.

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pAAV-CMV vector map

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pAAV-CMV vector map.

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Preparation of AAV2 particles using the AAVpro Helper Free System (AAV2)

Preparation of AAV2 particles using the AAVpro Helper Free System (AAV2)
Preparation of AAV2 particles using the AAVpro Helper Free System (AAV2). HEK293T cells are co-transfected with the three plasmids (pHelper, pAAV-CMV, and pRC2-mi342) that encode all of the components necessary to product recombinant AAV2 particles. After transfection, particles can be extracted using included AAV Extraction Solution.

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Effect of miRNA-342 on AAV2 titer

Effect of miRNA-342 on AAV2 titer
Effect of miRNA-342 on AAV2 titer. HEK293 cells were transfected with the three plasmids in the AAVpro Helper Free System (AAV2). AAV2 particles were extracted from virus-producing cells either expressing hsa-miR-342 (pRC2-mi342) or not (pRC2). Viral titer was evaluated using real-time PCR (total vector genome; vg). Expression of hsa-miR-342 in this system resulted in a two-fold increase in titer as compared to cells that did not express the microRNA.
6231 pAAV-ZsGreen1 Vector 20 ug USD $586.00

pAAV-ZsGreen1 Vector is an adeno-associated virus (AAV) vector plasmid that has terminal hairpin structures called inverted terminal repeats (ITRs) at both ends, which function as origins of genomic replication and contribute to packaging of viral particles. This vector plasmid allows you to produce high titer recombinant AAV vector together with AAVpro Helper Free System (AAV2) (Cat. #6230). It carries the ZsGreen1 gene, a variant of the reef coral Zoanthus sp. green fluorescent protein, and allows ZsGreen1 to be used as an indicator of transfection efficiency and transduction efficiency.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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pAAV-ZsGreen1 vector map

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6231: pAAV-ZsGreen1 Vector

6231: pAAV-ZsGreen1 Vector
6232 AAVpro® Purification Kit (AAV2) 2 Preps USD $434.00

License Statement

ID Number  
M84 This product is the subject of the claims of US patent No. 10072250 and its foreign counterparts.
M83 This product is the subject of the claims of US patent No. 9422576 and its foreign counterparts.

The purity and titer of the AAV vector is an important for achieving stable and effective introduction of genes into individual animals and cultured cells. Methods such as CsCl concentration gradient ultracentrifugation and iodixanol ultracentrifugation are commonly used to purify AAV2 vectors, but such methods are time-consuming and require careful technique to obtain high yields. The AAVpro Purification Kit (AAV2) allows for simple and fast (~4 hours) AAV2 particle purification from AAV2-producing cells.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Overview of AAV2 purification using the AAVpro Purification Kit (AAV2)

Overview of AAV2 purification using the AAVpro Purification Kit (AAV2)
Overview of AAV2 purification using the AAVpro Purification Kit (AAV2).

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Recovery, purity, and infectivity of purified AAV2 particles

Recovery, purity, and infectivity of purified AAV2 particles

Recovery, purity, and infectivity of purified AAV2 particles . AAV2 particles were purified with each company’s purification kit from virus-producing cells. The yield of each purified AAV2 vector was evaluated by real-time PCR (Panel A), and the purity of each purified AAV2 vector was evaluated by SDS-PAGE using 1 x 109 vg per lane (Panel B). Infectivity was measured by infection of HT1080 cells with purified AAV2 particles at 2,500 vg/cell and subsequent measurement of ZsGreen expression (Panel C).

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6232: AAVpro Purification Kit (AAV2)

6232: AAVpro Purification Kit (AAV2)
6666 AAVpro® Purification Kit Maxi (All Serotypes) 4 Preps USD $782.00

License Statement

ID Number  
M88 This product is covered by the claims of US Patent No. 10415020.
M84 This product is the subject of the claims of US patent No. 10072250 and its foreign counterparts.
M5 This product is covered by the claims of JP Patent No. 4857260.

The AAVpro Purification Kit Maxi (All Serotypes) is a four-prep kit that can be used for purifying AAV of any serotype without the need for ultracentrifugation. The purification yields highly pure AAV particles that can be used directly for in vivo transduction or transduction of cultured cells. Cryonase Cold-Active Nuclease is included. This product allows for purification of four virus preps, each from a culture size of 5 x T225 flasks.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Summary of the AAV particle purification protocol using the AAVpro Purification Kit Maxi (All Serotypes)

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Purity of viral particles collected using the AAVpro Purification Kit Maxi (All Serotypes)

Purity of viral particles collected using the AAVpro Purification Kit Maxi (All Serotypes)

Purity of viral particles collected using the AAVpro Purification Kit Maxi (All Serotypes). AAV viral particles of each serotype carrying the fluorescent protein ZsGreen1 were prepared from producer cells cultured in five T225 flasks. The AAV solution was purified using this product, and viral titer was measured using the AAVpro Titration Kit (for Real Time PCR) Ver. 2. SDS-PAGE was performed on each sample, with 1 x 109 vector genomes loaded per lane. For all serotypes, the AAV capsid proteins (VP1, VP2, and VP3) were observed to be the major bands present, confirming the purity of the AAV viral particles.

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Infection capacity of AAV particles purified with the AAVpro Purification Kit Maxi (All Serotypes)

Infection capacity of AAV particles purified with the AAVpro Purification Kit Maxi (All Serotypes)

Infection capacity of AAV particles purified with the AAVpro Purification Kit Maxi (All Serotypes). Infectivities of AAV particles of various serotypes were evaluated. Purified AAV particles carrying the fluorescent protein ZsGreen1 were used for infection 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. Results indicate the AAV particles purified using this kit were able to infect the cell lines tested.

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6666: AAVpro Purification Kit Maxi (All Serotypes)

6666: AAVpro Purification Kit Maxi (All Serotypes)


AAV FAQ

Adeno-associated virus FAQs

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