Viral Manipulation of Neural Stem/Precursor Cells - Viral Vector Approaches in Neurobiology and Brain Diseases - page 270

4. Maintain the slices in culture at 34 °C at the interface between
culture medium and a 5 % CO 2 atm, changing medium twice
a week;
5. The viral particles were produced as shown in Sect. 14.3.2 ;
6. After 2 weeks, inoculate viral particles in the suprapyramidal
region of the granule cell layer of the hippocampus. Viral par-
ticles (5 × 10 5 TU/ml) are microapplied to slice cultures using
a manual micromanipulator placed on a vibrationless table;
7. After 2-4 weeks, the slices can be processed for further
analyses.

3.6 In Vivo
Transduction of NPCs

The following protocol refers to stereotaxic injection of viral par-
ticles in adult mice SVZ ( see Note 17 ).

1. Weigh the animal and calculate the appropriate dose of anes-
thesia. Deeply anesthetize the animal with an appropriate
injectable (e.g., intraperitoneal injection of ketamine 100 mg/
kg and xylazine 10 mg/kg) or breathable (e.g., isoflurane)
anesthesia ( see Note 14 ). Shave the fur on the skull and clean
the skin with 70 % ethanol or another disinfectant;
2. Securely position in a stereotactic apparatus using bite and the
ear bars. With a scalpel, cut the skin making a midline incision
on the upper part of the head and separate subcutaneous tis-
sue, exposing the skull;
3. Measure the coordinates of the injection site of interest from
the bregma, as determined from a stereotaxic brain atlas and
mark the point of injection. Stereotactic coordinates for SVZ
starting from the dura: anteroposterior 0.6 mm, mediolateral
1.6 mm, and dorsoventral 3-2 mm;
4. Using a handheld drill with an appropriate tip, make a hole on
the surface of the skull, taking care not to cause injury to the
brain parenchima;
5. The viral particles are produced as shown in Sect. 14.3.2 ;
6. Inject the SVZ using a 33 G Hamilton (or similar) syringe
with 1 μl of recombinant viral particles (10 9 -10 10 TU/ml)
supplemented with Polybrene (4 μg/ml), at a rate of 0.25 μl/
min via a nanoinjector pump ( see Note 18 );
7. Wait 3-5 min before extracting the needle, and then slowly
withdraw it to avoid backflow of the virus;
8. Suture the mouse skin with absorbable suture and keep the
animal warm in an individual cage until it fully recovers
(Fig. 2 ).