Application of Viral Vectors to Motor Neuron Disorders - Viral Vector Approaches in Neurobiology and Brain Diseases - page 230

12. AAV serotype 6 effi ciently infects HEK 293 T cells. However,
the level of transduction highly depends on the serotype used
and should be carefully assessed before selecting the adequate
cell type to determine vector infectivity. It is not possible to
compare vector infectivity across various serotypes because of
the observed differences in tropism for target cells.
13. The infectivity is measured when the maximal level of trans-
gene expression is reached in the target cells, which can be
easily monitored by fl uorescence microscopy following trans-
duction with a GFP-expressing vector. The peak in expression
is observed at 48 h following infection of HEK 293 T cells
with AAV6 vectors. However, the kinetic of expression should
be assessed for each cell type and AAV serotype.
14. In HEK 293 T cells, the cmv and cba promoters drive trans-
gene expression at high and therefore easily detectable levels.
15. The concentration and incubation time of the S1 nuclease are
determined to effectively eliminate single-stranded DNA and
let most of the double-stranded DNA undigested. Optimization
can be made by comparing the abundance of single-stranded
(vector genome) versus double-stranded DNA (cellular
genome) before and after the reaction.
16. Contaminants might interfere with infectivity of injected par-
ticles and induce an undesired infl ammatory response.
17. Between P1 and P3, the skin of the pup is soft enough to be
pierced through by the needle. Moreover, the vector can easily
diffuse through the entire muscles of mouse neonates follow-
ing a single injection. Hence, no incision is required and intra-
muscular injections are performed directly through the skin.
18. The described procedure is suited for ICV injections in new-
born mice up to P4. Until that age, the skin and bone are
transparent enough to allow adequate landmark identifi cation.
19. A 3 × 3 × 3 mm polystyrene cube can be added at the base of
the needle to avoid penetrating too deep in the brain
parenchyma.
20. Bolus injection is critical to allow rapid diffusion of AAV6 par-
ticles in the CSF and subsequent infection of motor neurons
along the whole spinal cord axis. Slower injections might lead
to reduced diffusion and limit infection to motor neurons of
the brainstem and upper cervical spinal cord.
21. The Fast Green added to the virus preparation should make
the ventricles appear denser against a light source if the injec-
tion was successful.