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. 2011 Oct:Chapter 4:Unit 4.17.
doi: 10.1002/0471142301.ns0417s57.

Production of recombinant adeno-associated viral vectors and use in in vitro and in vivo administration

Steven J Gray  1 , Vivian W Choi, Aravind Asokan, Rebecca A Haberman, Thomas J McCown, Richard Jude Samulski
Affiliations

Production of recombinant adeno-associated viral vectors and use in in vitro and in vivo administration

Steven J Gray et al. Curr Protoc Neurosci. 2011 Oct.

Abstract

Adeno-associated virus is a nonpathogenic human virus that has been developed into a gene-delivery vector due to its high efficiency of infection for many different cell types and its ability to persist and lead to long-term gene expression. This unit describes efficient methods to generate high-titer, research-grade, adenovirus-free recombinant single-stranded and self-complementary adeno-associated virus in various serotypes, along with methods to quantify the viral vectors. Two detailed methods are provided for viral vector delivery into the rodent brain and spinal cord, and for histological detection of transgene expression of GFP.

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Figures

Figure 4.17.1
Figure 4.17.1
Generation of adenovirus-free recombinant adeno-associated virus. 293 cells (which supply the Ad E1 gene) are transfected using three plasmids: the plasmid carrying the transgene (sub201-gene “X”, AAV vector), the plasmid supplying the replication and capsid genes of AAV2 without terminal repeats (pXX2, AAV helper), and the plasmid supplying the adenovirus helper genes E2, E4, and VA RNA genes (pXX6, AD DNA), thereby generating Ad-free rAAV.
Figure 4.17.2
Figure 4.17.2
(A) pSub201. The plasmid contains the terminal repeats and the replication (rep) and capsid (cap) genes of AAV Type 2. The rep-cap fragment can be replaced by the gene cassette of interest, as only the terminal repeats are needed for packaging. (B) Plasmid pHpa-trs-SK.
Figure 4.17.2
Figure 4.17.2
(A) pSub201. The plasmid contains the terminal repeats and the replication (rep) and capsid (cap) genes of AAV Type 2. The rep-cap fragment can be replaced by the gene cassette of interest, as only the terminal repeats are needed for packaging. (B) Plasmid pHpa-trs-SK.
Figure 4.17.3
Figure 4.17.3
CsCl step gradient before and after centrifugation. The step gradient is generated by underlying the virus-containing clarified cell lysate with 1.3 and 1.5 g/m3 CsCl solutions (see Basic Protocol 1 for instructions on forming the gradient). After centrifugation, the virus resides at the interface between the 1.3 and 1.5 g/m3 CsCl solutions. Care should be taken when removing this layer not to remove any of the less-dense cellular debris that remains above the 1.5 g/m3 solution.
Figure 4.17.4
Figure 4.17.4
Green fluorescent protein (GFP) expression in the inferior colliculus and the globus pallidus 2 weeks after infusion of 1 μl of AAV-GFP virus.
See this image and copyright information in PMC

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