Gene Therapy for Chronic Pain: How to Manipulate and Unravel Pain Control Circuits from the Brain? - Gene Delivery and Therapy for Neurological Disorders - page 329

7. Titrate these positive samples following the procedure in
Sect. 3.1.2 .
8. Carry out two additional rounds of limiting dilution using the
stock of virus stored at −80 °C, as in steps 1-7 above.
9. The virus stock from one positive well selected and stored at
the end of the third round is used to expand the titer and
extract viral DNA ( see Note 1 ) in order to confi rm the pres-
ence of the inserted cassette by PCR.

1. Set up 3 T-225 fl asks with 7B cells (6 ml of cell suspension
resuspended in 60 ml of culture medium per fl ask).
2. The next day, add 0.75 × 10 6 PFU of the recombinant virus in
11 ml of culture medium. Aspirate the culture medium of the
3 T-225 fl asks (80-90 % confl uent), and add 3.5 ml of recom-
binant virus solution. Incubate for 1 h in the CO 2 incubator.
3. Aspirate the viral inoculum. Add 70 ml of culture medium per
fl ask and incubate for 48 h.
4. Harvest cells. Transfer medium and cells to a sterile 250 ml
centrifuge bottle, and centrifuge at 10,000 rpm with Sorvall
SLA-1500 or GSA rotor for 10 min.
5. Aspirate supernatant. Resuspend the cell pellets in 4 ml of cold
culture medium. Transfer to a 15-ml centrifuge tube.
6. Freeze-thaw three times, and after the fi nal thaw, sonicate for
∼

3.1.4 Virus Stock
Preparation with Sucrose
Gradient Purifi cation

15 s on setting 3 of a cup-horn sonicator (Branson Sonifi er
450). Add 9 ml of cold culture medium and centrifuge at full
speed in a benchtop centrifuge for 10 min.
7. Transfer supernatant to a 50-ml Sorvall tube containing 15 ml
30 % sucrose/PBS-D. Centrifuge at 18,000 rpm (38,000 × g )
for 2 h at 4 °C in Sorvall SS-34 rotor.
8. Carefully and thoroughly aspirate the supernatant. Resuspend
the pellet in 10 % sucrose/PBS-D, using 450 µl per T-225 fl ask
of cells. Pipette 100 µl aliquots and store at −80 °C.
9. Titrate following the procedure following the procedure in
Sect. 3.1.2 .

The HSV-1 vectors were stereotaxically injected in two DRt ros-
trocaudal parts of the left DRt following the coordinates of Paxinos
and Watson [ 14 ] and using the interaural line as a reference to
calculate the coordinates (Table 1 ). The procedure for HSV-1-
vector injection is performed on Wistar rats (Charles River, Spain)
weighing 285-315 g as follows:

3.2 Stereotaxic
Injection of HSV-1
Vectors
and Immunodetection
of Transduced
Neurons

1. Anesthetize the animals with an intraperitoneal (i.p.) injection of
a mixture of ketamine hydrochloride (0.06 g/kg) and medeto-
midine (0.25 g/kg), and place them on a stereotaxic frame
(David Kopf Instruments; Tujunga, CA, USA) by positioning

3.2.1 Stereotaxic
Injections