Gene Delivery and Gene Therapy for Alzheimer’s Disease - Gene Delivery and Therapy for Neurological Disorders - page 105

Mix DNAs with water and then CaCl 2

TUBE 2 mixture Per T150 fl ask For ten fl asks

2× HBS 1200 µl 12,000 µl

(c) Slowly add Tube 1 mixture into Tube 2 in a dropwise
manner using a pipette with vortex in hood.
(d) Incubate this combined solution in the hood at room tem-
perature for 30 min.
(e) Vortex the combined solution again and immediately add in
a dropwise manner into the T150 fl ask. Incubate for 24 h.

5. 24 h after transfection, change media in the hood: remove all
media and replace with 25 ml of fresh cDMEM media.
6. 48 h after transfection, collect the cells and media from each
fl ask using a cell scraper and transfer to a 250 ml conical tube.
7. Centrifuge the tube at 300 × g , 4 °C, 20 min.
8. Discard the supernatant and move on the next step ( see Note 2 ).

Resuspend the pellet in 7 ml cell suspension buffer ( see Table 3 )
and vortex.

3.2.2 Crude AAV Particle
Extraction

1. Freeze the cell suspension in dry ice/95 % methanol.
2. Thaw at 37 °C in water bath, and vortex.
3. Repeat the freeze-thaw cycle of cell suspension a total of four
times. This will lyse cells by swelling and breakage due to crys-
tal formation. The suspension can be stored at −80 °C after
fi nal freezing step.
4. Centrifuge at maximum speed, 4 °C, for 15 min.
5. Save the supernatants in a new 50 ml conical tube, and discard
the pellet.
6. Add 7 µl Benzonase (fi nal concentration: 50 U/ml), mix to
remove nucleic acid contaminants, and incubate at 37 °C for
1 h.
7. After Benzonase treatment, add 23 ml 50 mM cell suspension
buffer.

1. Prepare the iodixanol solutions ( see Table 2 ):
60 % iodixanol: original solution
40 % iodixanol: original solution 20 ml + PBS-MK 10 ml
25 % iodixanol: original solution 20 ml + PBS-MK 28 ml
15 % iodixanol: original solution 12 ml + PBS-MK 21.6 ml,
+2.5 M NaCl 14.4 ml

3.2.3 Iodixanol Gradient
Purifi cation (See Note 3 )