Adeno-Associated Virus-Based Vectors - Viral Vector Approaches in Neurobiology and Brain Diseases - page 41

In the following protocol, transducing efficiency is assessed on
HeLa cells infected with a GFP-expressing rAAV-2 vector.
The day before infection seed 2 × 10 5 HeLa cells in a 24-well plate
in 0.5 ml of DMEM supplemented with 10 % of FBS.
Incubate cells overnight at 37 °C.
On the day of infection, prepare a series of tenfold dilutions of the
rAAV stock as described for the infectious center assay.
Add 100 μl of serially diluted rAAV to each well. Include a
mock-infected control. Perform each sample and control in
duplicate.
Incubate cells at 37 °C for 48-72 h to allow gene expression.
Add 0.2 ml of trypsin to each well. Incubate cells at 37 °C until
they have detached. Transfer cells and medium into a micro-
centrifuge tube.
Centrifuge at 1,000 rpm for 5 min. Wash cell pellet with 1 ml
of PBS.
Repeat the wash step one more time and keep cells on ice.
Determine the percentage of GFP-expressing cells by flow cytometry
using CellQuest software.
Calculate the transducing titer in transducing units per ml
(TU/ml) using the rAAV dilution that yields approximately
20 % of GFP-positive cells.
Transducing titer: number of fluorescent cells × dilution
factor × 1,000

3.5 Vector Quality
Control

(a) Assay for protein purity
Silver-stained or krypton infrared protein-stained SDS-
polyacrylamide gels can be used to assess the purity of a
vector preparation and to determine the ratio of VP proteins
versus contaminating proteins. The three AAV capsid proteins
VP1, VP2, and VP3 should be detected in an approximate
stoichiometric ratio of 1:1:10 with correct molecular weights
of 87, 72, and 62 kDa, respectively.
Add 2 μl of 6× Laemmli buffer to 10 μl of viral sample.
Denature for 5 min at 100 °C. Load samples and protein
ladder (1 μl) on a 10 % SDS-polyacrylamide gel. Run samples
in 1× Tris-Glycine, 0.1 % SDS. Run gel at 100 V until dye has
reached the bottom of the gel.
Proceed to staining using the krypton infrared protein stain kit as
briefly described below.
Place the gel in a clean tray with sufficient volume of fixing solu-
tion (50 % ethanol and 15 % acetic acid). Agitate for 10 min.
Decant the gel fixing solution. Add fresh fixing solution. Agitate
for 10 min.