Yonsei Med J.  2016 May;57(3):790-794. 10.3349/ymj.2016.57.3.790.

Simple Purification of Adeno-Associated Virus-DJ for Liver-Specific Gene Expression

• Liu J
• Moon YA

• KMID: 2374103
• DOI: http://doi.org/10.3349/ymj.2016.57.3.790

Abstract

Recombinant gene expression using adeno-associated viruses (AAVs) has become a valuable tool in animal studies, as they mediate safe expression of transduced genes for several months. The liver is a major organ of metabolism, and liver-specific expression of a gene can be an invaluable tool for metabolic studies. AAV-DJ is a recombinant AAV generated by the gene shuffling of various AAV serotypes and shares characteristics of AAV2 and AAV8. AAV-DJ contains a heparin-binding domain in its capsid, which suggests that a heparin column could be used for the purification of the AAV. Given that AAV-DJ has been only recently available, relatively little is known about the optimal preparation/purification and application of AAV-DJ. Here, we present a simple large-scale preparation method that can generate 3×10(13) viral particles for in vivo experiments and demonstrate liver-specific gene expression via systemic injection in mice.

Keyword

AAV-DJ; large scale production; liver-specific expression

MeSH Terms

Animals
Capsid
Capsid Proteins/*genetics
Dependovirus/*genetics
*Gene Expression
*Genetic Vectors
Genome, Viral/genetics
Hep G2 Cells
Humans
Liver/*metabolism
Mice
Mice, Inbred C57BL
Capsid Proteins

Figure

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  Fig. 1 Generation of AAV-CAG-GFP. GFP was cloned into pAAV-CAG vector that has chicken actin promoter and designated as pAAV-CAG-GFP. Empty pAAV-CAG was used as a control (pAAV-control). pAAV-CAG-GFP or pAAV-control was co-transfected with pDJ and pHelper to QBI-HEK 293A cells to generate AAV-DJ-GFP or AAV-DJ-control, respectively. GFP, green fluorescent protein.

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  Fig. 2 Flow chart of AAV-DJ purification.

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  Fig. 3 Liver specific expression of GFP using AAV-DJ-GFP. AAV-DJ-control and AAV-DJ-GFP were injected to mice via tail vein (1012 viral particles/mouse). After 3 months, organs indicated in the figure were harvested and the expression of GFP protein was detected using an IVIS scanner. GFP, green fluorescent protein.

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  Fig. 4 Dose dependent expression of GFP using AAV-CAG-GFP in mice. AAV-CAG-GFP virus was injected at doses of 1011, 3×1011, and 1012 viral particles/mouse. Three weeks after injection, liver was harvested and GFP expression was observed under a fluorescent microscope. GFP, green fluorescent protein.

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