Gene Therapy for Epilepsies - Gene Delivery and Therapy for Neurological Disorders - page 247

Scissors (for cutting nichrome wires)
Soldering iron or workstation
Solder
Sockets (e.g., stainless steel gold-plated socket, Plastics One
Inc., Roanoke, VA, USA)
Superglue
Stainless cannula (e.g., Product C313G, Plastics One Inc.)
Stereotaxic electrode/cannula holder
Stainless steel mounting screws (e.g., Product 0-80 X 3/32,
Plastics One Inc.)
Stainless screw with electrode (e.g., Product E363/20/2.4/
SP, Plastics One Inc.)
Acrylic dental cement
26-G needle
6-channel plastic electrode pedestal (e.g., Product MS363,
Plastics One Inc.)
Dust cap (e.g., Product 363 DC, Plastics One Inc.)
Cannula dummy (e.g., Product 313 DC/1, Plastics One Inc.)
Internal cannula (or injector cannula, e.g., Product C313I/SP
with custom guide length, Plastics One Inc.)
electroencephalogram (EEG) system
Connector cable to the EEG (e.g., Product 363-340/6,
Plastics One Inc.)
Transparent Plexiglas circular enclosure of approximately
50-cm diameter, 60-cm height (this is for rats, smaller one can be
used for mice)
Video recording device
Mirror
Kainic acid
PE50 polyethylene tubing
10-μl Hamilton syringe
Pentobarbital
0.5-cc insulin syringe, with 28-G needle

3 Methods

Since for the majority of preclinical studies and possibly the even-
tual clinical application of epilepsy gene therapy, strong and
widespread vector transduction is desired; we will describe the
method for AAV1 vector production.
We prepare vectors in batches of five 15-cm dishes, which in
our experience will provide sufficient viral particles to generate a
vector stock of high genomic titer. The general procedure of
recombinant AAV production is outlined in Fig. 1 .

3.1 AAV Vector
Production

1. Plate out HEK293 cells 24 h prior to transfection at 2 × 10 7 cells
per 15-cm dish in 20 ml of complete DMEM/10 % FBS to
achieve 70-80 % confluency at time of transfection. Make sure
the cells are well mixed and evenly distributed over the dish.