Lentiviral Vectors in Huntington’s Disease Research and Therapy - Viral Vector Approaches in Neurobiology and Brain Diseases - page 202

Fig. 8 Expression of the mutant Htt gene in primary cultures of striatal neurons.
Detection of nuclear ( indicated by V ) and neuritic mutant Htt inclusions 8 weeks
postinfection, with LV-VSV-G encoding Htt171-82Q [ 32 ]

3.9 Culture
and Transduction
of Primary Cultures
of Cortical Astrocytes

Primary cultures of cortical astrocytes are generated from P1/P2
newborn C57/Bl6 mice. Forebrains are removed aseptically from
the skull and placed in ice-cold PBS. The meninges are carefully
removed and the neocortex is dissected free from the brainstem,
thalamus, striatum, and hippocampus. Cells are then dissociated
with a 5-ml pipette and passed through needles of decreasing
gauge from 20 to 21 gauge. Cells are used to seed DMEM
supplemented with 10 % FBS, 0.5 mM glutamine, and 0.1 % strep-
tomycin/penicillin (10

3.9.1 Primary Culture
of Cortical Astrocytes

μ

g/ml and 10 U/ml, respectively) in
6-well plates. The medium is replaced completely every 2 days.
Primary astrocytes are maintained in culture for up to 3 weeks.
They reach maximum confl uence after 12-15 days.

The equivalent of 10 ng of p24 antigen/10 5 cells (i.e., 180-200 ng
of p24 antigen of virus/well for a 6-well plate and 35-40 ng of p24
antigen of virus/well for a 24-well plate) of LV-MOK-G-miRT is
used to transduce cortical astrocytes. Transduction should be car-
ried out after 10-11 DIV to ensure that cell confl uence is optimal.
Transgene expression is visible 2 days after transduction and expres-
sion is maximal 11 days after transduction (i.e., around 21 DIV).

3.9.2 Transduction of
Primary Cortical Astrocytes

3.10 In Vivo
Transduction: HD
Rodent Model and LV
Tropism

LV tropism can be investigated in primary cultures in vitro.
However, one important limitation is that the tropism observed
may differ from that observed in vivo. Indeed, whereas MOK-G
confers an astrocytic tropism on LVs in vivo; LVs pseudotyped
with MOK-G transduce both astrocytes and neurons in vitro (data
not shown). It is therefore important to check the tropism of LVs
by stereotaxic injection in rodent brain.
Adult rats weighing 200-250 g and adult mice weighing
20-25 g are anesthetized with a ketamine/xylazine solution:

3.10.1 Stereotaxic
Injection: Standard
Protocol and HD Model
In Vivo