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.

Adeno-associated virus is a nonpathogenic human virus that has been developed into a gene-delivery vector due to its high efficiency of infection in many different cell types and its ability to persist and lead to long-term gene expression. The vector is also a valuable tool in molecular biology experiments. 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.

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.

Recombinant adeno-associated viral (rAAV) vectors are capable of delivering genes to both dividing and nondividing cells. This property, along with the physical stability of the virions, permits transduction of cells both in tissue culture and in the whole animal. In this unit, protocols for the adenovirus-free production of rAAV vectors, their purification, and their quantitation are provided. The production and purification of the rAAV vectors are described, using two different methods (see Basic Protocol and Alternate Protocol). Two assays for determining rAAV titers are also presented: dot blot (see Support Protocol 1) and in vitro infection with a transgene expression assay (see Support Protocol 2).An overview of the rAAV production procedure is shown in Figure 12.9.1. The production of adenovirus-free rAAV particles requires four elements: an rAAV vector plasmid containing the transgene flanked by AAV inverted terminal repeats (ITRs), a plasmid that supplies the AAV viral proteins necessary for replicating and packaging the rAAV sequences (AAV helper plasmid), a plasmid supplying the adenoviral helper genes (Ad helper plasmid), and tissue culture cells. After cotransfection of the plasmids into the tissue culture cells, rAAV is produced and the cells are harvested. The rAAV is then purified either on CsCl gradients or on heparin sepharose columns and dialyzed for storage or before use in animals.

Adeno‐associated virus (AAV) vectors are able to transduce CNS cells in vitro and in vivo for extended periods of time without immune complications. The ability to efficiently deliver transgenes in vivo with recombinant AAV makes this an ideal vector for neuronal gene therapy. This unit describes the production of recombinant AAV vectors and presents a protocol for infusing these viruses into the brains of intact animals.