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In-Depth Information
[
61
]. Several studies showed contrasting efficiencies of NPC trans-
duction with adenoviral vectors [
66
-
69
]. Falk et al., for example,
tested gene delivery to adult mouse NPCs using viral and nonviral
vectors and found that the efficiency of adenoviral gene delivery in
comparison to other nonviral and viral vectors was the highest
[
68
]. In contrast, Schmidt et al. showed that adult mouse NPCs
are not permissive for AdH5 mediated gene transfer due to a lack
of expression of appropriate receptors on NPC plasma membrane
[
70
]. However, subsequent studies showed that it is possible to
increase transduction efficiency of adenoviral vectors by engineer-
ing the capside proteins to bind NPC-specific peptides [
71
,
72
].
Adeno-Associated Viruses (AAVs)
are unable to establish an
infection without helper viruses and are not known to cause pathol-
ogy in humans. They have broad host and cell tropism and trans-
duce both dividing and nondividing cells [
73
]. Importantly, wild
type AAV integrate into chromosome 19, allowing stable expres-
sion of the transgene [
61
]. However, they can carry inserts with a
maximum length of 4 kb. The serotype capsid of AAV plays an
important role in determining the specificity of infection. The
serotypes that preferentially transduce astrocytes and NPCs are
AAV4, AAV5, and AAV9 [
74
-
76
].
In this chapter, we will specifically focus on the protocols
regarding the use of lentiviruses to transduce rodent adult NPCs,
but similar approaches can be performed also using other virus
backbones or on different cell sources (e.g. human).
We will describe first the protocols for the derivation and expan-
sion of NPC lines from the adult mouse SVZ, and those for the
production and the titration of the lentiviral particles, using a third
generation packaging system in the 293 T cell line (Sects.
14.3.1
and
14.3.2
); then these particles can be used to infect:
1. NPCs for in vitro studies or for transplantation in animal mod-
els, as described in Sects.
14.3.3
and
14.3.4
;
2. Organotypic slice cultures in Sect.
14.3.5
, as they represent a
widely accepted experimental model to replace or reduce ani-
mal experiments and have been increasingly used in molecular
biology. Organotypic slices are viable for as long as 20 weeks
in culture offering the possibility to study a variety of mecha-
nisms and treatment strategies for neurodegenerative disor-
ders over time [
77
-
85
];
3. Direct lentiviral injection in vivo into the SVZ or the SGZ, as
shown in Sect.
14.3.6
.
The combination of the NPCs as a cellular system ready to be
improved with a versatile tool such as the viral vectors has permit-
ted (and will allow further in the future) an impressive advance in
the knowledge of neurobiology and in the field of regenerative
medicine as a new strategy for brain repair.
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