Gene Therapy for Huntington’s Disease - Gene Delivery and Therapy for Neurological Disorders - page 136

Table 2
Solutions and reagents

Solutions

Volume Reagent Final concentration

Lysis buffer — heparin

15 mL 5 M NaCl 150 mM

10 mL 1 M Tris, pH 8 20 mM

Make up to a final volume of 500 mL with ultrapure water and adjust pH to 8 with HCl. Filter
sterilize and store at 4 °C

Lysis buffer — iodixanol

7.5 mL 5 M NaCl 150 mM

10 mL 1 M Tris, pH 8.5 50 mM

Make up to a final volume of 250 mL with ultrapure water and adjust pH to 8.5 with HCl. Filter
sterilize and store at 4 °C

10 % sodium deoxycholate

0.5 g Sodium deoxycholate (Sigma, #D6750) 10 %

Dissolve in ultrapure water to a final volume of 5 mL and filter sterilize. Prepare fresh solution as
required

Reagent Supplier Item number

Benzonase Sigma E1014-25KU

Our standard preparation of AAV vector typically yields
~300 μL of AAV vector of 10 12 -10 13 vector genomes/mL from
5 × 15 cm tissue culture plates.

Human embryonic kidney 293 (HEK293) cells (low passage
HEK293 cells, Microbix Biosystems Inc., Ontario, Canada) are
cultured in complete DMEM in T175 cm 2 tissue culture flasks.

3.1.1 HEK293 Cell

Culture

1. For each batch of rAAV vector, HEK293 cells are seeded into
5 × 15 cm diameter tissue culture plates in 25 mL complete
DMEM. The number of cells in one fully confluent T175 flask is
sufficient to seed 3.5-3.75 15 cm plates, such that the plates are
70 % confluent at the time of transfection ( see Notes 2 and 3 ).
2. The following day, 3 h prior to transfection, remove the media
from each plate and replace with 25 mL IMDM.
3. Bring transfection reagents to room temperature, and immedi-
ately prior to transfection, prepare the DNA mix in a 50 mL
tube as follows:

3.1.2 Transfection
of HEK293 Cells