Adeno-Associated Virus-Based Vectors - Viral Vector Approaches in Neurobiology and Brain Diseases - page 40

microcentrifuge tube. Rinse each well with 100 μl of PBS
and transfer into the same microcentrifuge tube.
Prepare the replication center assay apparatus with two pieces of
Whatman paper prewetted in PBS. Soak the nylon membrane
in PBS and place on top of the Whatman paper.
Apply 5 ml of PBS to each filter. Apply the cell suspension drop
wise into the PBS buffer on top of the nylon membrane.
Gently rock for even distribution. Apply vacuum.
Carefully remove the nylon membranes from the manifold.
Transfer the nylon membranes with cell side up on a piece of
Whatman paper prewetted in denaturing solution. Denature
for 5 min.
Transfer the nylon membranes on a piece of Whatman paper
prewetted in neutralizing solution. Neutralize for 10 min.
Transfer the nylon membranes on a piece of Whatman paper
prewetted in 2× SSC and rinse for 2 min.
Air-dry the nylon membranes for 5 min.
Immobilize DNA on membrane using a UV crosslinker setup at
1,200 × 100 μJ/cm 2 .
Place the membranes in a large hybridization bottle containing
20 ml of 0.75× NW buffer. Prehybridize at 65 °C for at least
2 h in a hybridization incubator.
Label a rAAV-specific 32 P-probe, hybridize, and wash filters as
described for the dot blot assay.
Place the membranes against an X-ray film and expose overnight at
room temperature.
Develop the film and count the number of black dots per mem-
brane; calculate the viral titer in infectious unit/ml (IU/ml):
Infectious titer: number of black dots × dilution factor × 1,000
(d) Transduction Assay
The transducing unit titer refers to the number of particles
capable of transgene expression in a given cell type. The
transduction assay is used to determine the expression of a
transgene that can easily be measured such as the green
fluorescent protein (GFP), secreted alkaline phosphatase
(SEAP), and β-galactosidase. Quantification of transducing
rAAV particles depends on the cell type and serotype used,
the promoter driving transgene expression, and the transgene
by itself. It is therefore difficult to use this assay to compare
titers obtained from different combinations of serotypes and
transgene cassettes. The adenovirus can also be used to
determine the transducing titer as it can greatly enhance
rAAV transduction efficiency.