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

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.

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.

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.

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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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Notice to purchaser

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