Gene Delivery and Gene Therapy for Alzheimer’s Disease - Gene Delivery and Therapy for Neurological Disorders - page 100

for construction of AAV2-MCS-WPRE expression vectors carrying
the gene of interest, generation of adenovirus-free hybrid AAV2/1
vectors through cotransfection of helper plasmid, AAV1 and AAV2
constructs into AAV293 cells, purifi cation and tittering of rAAV,
stereotaxic injection into animals, and fi nally histological analysis
of gene expression.

2 Materials

1. For all tissue culture procedures, use standard tissue culture
incubator, with 5 % CO 2 humidifi ed environment at 37 °C.
A separate 37 °C incubator should be used for incubation of
bacteria, to avoid contamination.
2. The use of AAV-293 cells is highly recommended for viral
generation. The cells are derived from HEK293 cell line and
produce higher viral titers. AAV particles are produced by trans-
fection of the cells with three plasmids (an inverted terminal
repeat (ITR) sequences-containing plasmid, AAV replication
and capsid gene-containing plasmid, the early region 1 (E1)-
deleted adenoviral helper plasmid). The E1 gene is provided by
the AAV-293 cells in trans to produce infectious AAV particles,
and viral generation should be performed in a biosafety level 2
(BSL-2)-approved facility. Here we use a pAAV serotype 2
including cytomegalovirus/chicken -actin hybrid promoter,

multicloning sites and Woodchuck posttranscriptional regula-
tory element (pAAV2-MCS-WPRE), p5E18RXC1, and
pAd

ʲ

F6 (both provided from Dr. James Wilson, the University
of Pennsylvania Gene Therapy Program).

∆

3. In Tables 2 and 3 , we provide a list of all reagents and solutions
described in the Sect. 3 .

3 Methods

Recombinant AAV vectors are easily produced by exchanging the
whole viral genome except ITRs with any genomic DNA or com-
plementary DNA (cDNA) of interest. However, the DNA with all
essential elements for AAV generation must be less than 5.0 kb in
length because of the viral packaging limit. A standard procedure
of DNA subcloning is suffi cient for this purpose. Here we use a
pAAV2-MCS-WPRE as a cis -plasmid for AAV generation.

3.1 Generation
of rAAV Vectors

1. Obtain DNA fragments to be inserted into MCS of pAAV2-
MCS-WPRE containing ITRs, which will fl ank the gene of
interest. The DNA can be a proofreading PCR amplifi cation of
complementary DNA region or directly dissected from other
plasmids including cDNAs.

3.1.1 Subcloning
of Genome of Interest into
pAAV2-MCS-WPRE