Viral Manipulation of Neural Stem/Precursor Cells - Viral Vector Approaches in Neurobiology and Brain Diseases - page 265

contained in a brain slice of 2-3 mm, as such by means of a
mouse brain matrix that allows to consistently and reproduc-
ibly slice the mouse brain; coronal sections are taken 2 mm
from the anterior pole of the brain, excluding the optic tracts
and 3 mm posterior to the previous cut;
3. Isolate the SVZ of the lateral ventricles from the coronal sec-
tion using iridectomy scissors under a dissecting microscope
and pool tissues derived from at least two mice to generate
each culture;
4. Transfer the dissected tissue to a 15 ml tube with NPC diges-
tion Medium and incubate it for 20-30 min at 37 °C on a
rocking platform or in the incubator with occasional mix by
tube flicking;
5. At the end of the incubation, centrifuge the tube at 200 × g for
10-12 min, remove the supernatant, and mechanically triturate
the pellet in 2 ml of EBSS with a p1000 pipette ( see Note 1 );
6. Centrifuge the pellet at 200 × g for 12 min and then dissociate
it again with a p200 pipette and plate in a 25 cm 2 flask in the
medium for expansion of mouse NPCs ( see Note 2 );
7. After approximately 1 week, a small percentage of the isolated
cells start to proliferate, giving rise to small cellular aggregates,
which are similar to rounded spheres ( neurospheres ) and which
grow in suspension. When the neurospheres reach the neces-
sary dimension (about Ø ≥ 150-200 μm in diameter), harvest
the cells in a 15 ml tube using a sterile pipette and centrifuge
them at 50 × g for 10-12 min;
8. Remove the supernatant, add 200 μl of Accumax, and incu-
bate the cells at 37 °C for 10 min. Mechanically dissociate the
pellet (e.g., by forcing it few times through a sterile tip, until a
single cell suspension is obtained), count the number of viable
cells (e.g., by trypan blue exclusion), and plate them in an
appropriate cell culture flask ( see Note 3 );
9. Repeat this procedure at each passage of dissociation.

Lentiviral vectors are produced by transient transfection of the len-
tiviral vector backbone carrying the coding sequence of interest
(transfer vector) and the packaging plasmids in HEK293T cells.
The protocol described below refers to the production of a third
generation, self-inactivating, replication defective lentiviral vector
obtained by transfection with the Calcium Phosphate precipitate
method. Other methods can be used to transfect the packaging cell
lines ( see Note 4 ).

3.2 Lentivirus
Production and
Titration

3.2.1 Lentivirus
Production

1. Grow the HEK293T cells in IMDM supplemented with 10 %
of fetal bovine serum, ultra-glutamine, and antibiotics;
2. Approximately 24 h before the transfection, seed 9 × 10 6 cells/
dish in a final volume of 20 ml of complete IMDM in tissue