Application of Viral Vectors to Motor Neuron Disorders - Viral Vector Approaches in Neurobiology and Brain Diseases - page 219

(pAAV-cmv-GFP) in a polystyrene 50 ml Falcon tube.
Complete the volume to 5 ml with H 2 O. Add 5 ml of HBS
dropwise to the DNA/CaCl 2 solution, while vortexing gently.
Incubate 10 min at room temperature and distribute the trans-
fection mix in the cell factory. Six hours later, the medium is
replaced with serum-free Episerf medium ( see Note 6 ).
3. Vector harvesting . Two days after transfection, collect cells in
50 ml polypropylene Falcon tubes. Harvest the conditioned
Episerf culture medium. The cells are resuspended by trypsin-
ization using 10 ml of trypsin-EDTA per cell factory. Finally,
rinse the plate with 10 ml PBS and add the PBS to the tubes.
Centrifuge for 5 min at 1,500 rpm. Rinse the pellet once in
PBS. Finally, resuspend the cells in PBS (5 ml for 5 cell facto-
ries) and freeze the cell pellet at −20 °C.
4. Cell lysis . Lyse the cell pellet with three cycles of freezing
(12 min in dry ice mixed with 70 % ethanol) and thawing
(10 min in 37 °C water bath). Vortex after thawing. Add
50U/ml Benzonase and incubate 30 min at 37 °C. Add the
CHAPS detergent at a fi nal concentration of 0.5 % and incu-
bate for 30 min at 37 °C. Centrifuge 15 min at 3,000 rpm and
pass the supernatant through a 0.8 m fi lter ( see Note 7 ).

5. Purifi cation step 1: iodixanol gradient . A four-step gradient is
prepared in a 30 ml Optiseal tube. The tube is rinsed with PBS
and fi lled with viral lysate (volume

μ

6 ml). Next, four different
concentrations of iodixanol-containing Optiprep solution are
sequentially added at the bottom of the Optiseal tube using a
10 ml syringe connected to a stainless steel 18G needle
(1.27 × 89 mm). Layer 1: 7 ml of 15 % iodixanol (12.5 ml
Optiprep with 37.5 ml PBS-MK/1 M NaCl); layer 2: 5 ml of
25 % iodixanol (20.8 ml Optiprep with 29.2 ml PBS-MK and
125

≤

l 0.5 % Phenol red); layer 3: 5 ml of 40 % iodixanol
(33 ml Optiprep with 17 ml PBS-MK); layer 4: <9 ml of 60 %
iodixanol (20.8 ml Optiprep with 29.2 ml PBS-MK and 125

μ

l
0.5 % Phenol red). Centrifuge in 70Ti rotor at 65,000 rpm for
68 min at 4 °C.
Insert an 18G needle connected to a 10 ml syringe into
the lower portion of the 40 % iodixanol fraction where the
virus accumulates and carefully aspirate this clear fraction
(4-5 ml). Avoid collecting any contaminant proteins that form
a visible interphase between layers 2 and 3. Dilute 1:1 the col-
lected fraction with PBS pH 7.4.
6. Purifi cation step 2: heparin affi nity chromatography . Connect a
1 ml HiTrap Heparin column to a high performance liquid
chromatography system (HPLC) with a UV detector ( see
Notes 8 and 9). Stabilize the column with at least 50 ml of
bidistilled H 2 O at a fl ow rate of 5 ml/min. Sequentially wash
the column with each time 50 ml of PBS, PBS + 0.4 M NaCl

μ