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  • Customer data: Purified AAV9 delivery (mouse brain)
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Tech Note

Gene transduction of mouse visual cortex using AAV vector purified with the AAVpro Purification Kit Maxi (All Serotypes)

Data kindly provided by Dr. Masato Uemura, Department of Physiology, The University of Tokyo School of Medicine

Introduction Results Conclusions Methods

Introduction  

Adeno-associated viruses (AAVs) are commonly used for gene delivery in vivo, and our AAV purification kits power these experiments in laboratories around the world. The ability to maintain high purity and titer is critical for producing meaningful data. Along with the simplicity of single-kit precipitation and filtration steps, we provide you with the ability to purify AAV particles with any capsid.

In one example, Dr. Uemura, at The University of Tokyo School of Medicine, used one of our AAVpro "All Serotypes" purification kits to purify an AAV9 vector carrying GCaMP (a calcium-sensitive fluorescent protein) for a study of the mouse visual cortex.

Results  

Green fluorescence of GCaMP was observed broadly

Dr. Uemura produced GCaMP-carrying AAV9 vectors in HEK293 cells. Following purification using the AAVpro Purification Kit Maxi (All Serotypes), the AAV particles were injected into the mouse visual cortex. The images one week later revealed widespread fluorescence in vivo.

Fluorescence of GCaMP in mouse visual cortex. Bright field (A) and fluorescent (B) microscopy images from above a glass window approximately one week after AAV injection. (Scale bar, 1 mm).

A closer look revealed GCaMP localized in the soma

Successful AAV9-mediated delivery and expression was also evident at the cellular level. As demonstrated via two-photon microscopy, Dr. Uemura found GCaMP fluorescence localized in the soma.

Fluorescence of GCaMP at the cellular level. Two-photon microscopy images of the visual cortex at a depth of 200–300 µm. Single-scanned image (A) and the average of 600 frames over ~5 min (B). (Scale bar, 100 µm).

Conclusions  

AAV9 purified by the AAVpro "All Serotypes" kit delivered a functional GCaMP, under control of the Tet-Off system, when injected directly into the mouse visual cortex.

Methods  

AAV vector preparation

AAV9 vector carrying GCaMP under the control of the Tet-Off system was produced in HEK293 cells using the calcium phosphate transfection method. Cells cultured in seven 15-cm dishes were harvested to purify AAV particles using the AAVpro Purification Kit Maxi (All Serotypes) (Cat. # 6666). Dulbecco's phosphate-buffered saline was used in place of the kit's Suspension Buffer, and the AAV particles were concentrated to ~125 µl using an Amicon Ultra-4 Centrifugal Filter Unit* (Amicon, Cat. # UFC810008). Viral titer was measured by qPCR (~1 x 1014 GC/ml).

*The Amicon Ultra-4 Centrifugal Filter Unit, which has a typical final concentrate volume of 50 µl, is not a component of the AAVpro Purification Kit Maxi (All Serotypes) (Cat. # 6666). The kit includes the Amicon Ultra-15, 100 kDa Centrifugal Filter Unit, with a typical final concentrate volume of 200 µl.

Injection of the prepared AAV vector into mouse visual cortex

~800 nl of the purified AAV vector was injected into mouse visual cortex. Approximately one week after injection, GCaMP expression was observed in vivo using light microscopy oriented above a glass window covering the injected region. Fluorescence at the cellular level was observed at a 200–300 µm depth in the same region of the visual cortex using a two-photon microscope.

Related Products

Cat. # Product Size License Quantity Details
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.

Documents Components You May Also Like Image Data

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

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

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)
6675 AAVpro® Purification Kit Midi (All Serotypes) 4 Preps USD $332.00

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 Midi (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 1 x T225 flask.

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.

Documents Components Image Data

Back

Gene transduction of iPSC-derived cardiomyocytes using AAV1, 2, 5, and 6.

Gene transduction of iPSC-derived cardiomyocytes using AAV1, 2, 5, and 6.

Gene transduction of iPSC-derived cardiomyocytes using AAV1, 2, 5, and 6. AAV1, 2, 5, and 6 vectors carrying the ZsGreen1 gene were purified with the AAVpro Purification Kit Midi (All Serotypes) kit. 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.

Back

6675: AAVpro Purification Kit Midi (All Serotypes)

6675: AAVpro Purification Kit Midi (All Serotypes)
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.

Documents Components You May Also Like Image Data

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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)


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