Gene Delivery and Gene Therapy for Alzheimer’s Disease - Gene Delivery and Therapy for Neurological Disorders - page 104

Fig. 1 Schematic diagram of rAAV generation by cotransfection of AAV cis and trans vectors and adenovirus
helper vector into AAV-293 cells

2. Co-transfection of AAV1, AAV2-MCS-WPRE-[cDNA], and
pAd F6 into the AAV-293 by CaPO 4 method (Xiao et al.

[ 25 , 37 ]) to obtain AAV2/1 hybrid virus expressing gene of
interest

∆

Method:

1. Maintain AAV-293 cells in T150 fl asks with 25 ml cDMEM
( see Table 3 ) at 37 °C, 5 % CO 2 until they reach 80-90 %
confl uency.
2. One day before transfection, split the cells into three T150
fl asks to obtain 40-60 % confl uency on the day of transfection
(1:3 dilution), and incubate the cells until next day. We usually
prepare ten fl asks for one virus.
3. Three hours before transfection, change cDMEM media.
4. Prepare transfection mixture approximately 1 h before trans-
fection as follows:
(a) Thaw all transfection components (plasmid DNA solu-
tions, 2 M CaCl 2 , 2× HBS; see Table 3 ) to room tempera-
ture and mix.
(b) Make the following solution in a 50 ml conical tube:

TUBE 1 mixture Per T150 fl ask For ten fl asks

pAd ∆ F6 36 µg 360 µg

pAAV2-MCS-WPRE 12 µg 120 µg

p5E18RXC1 (AAV1) 12 µg 120 µg

Sterilized water to 1,050 µl 10,500 µl

2 M CaCl 2 150 µl 1,500 µl