Lentiviral Vectors in Huntington’s Disease Research and Therapy - Viral Vector Approaches in Neurobiology and Brain Diseases - page 201

Table 3
Cell density required for striatal primary culture

Area
of 1 well

Neuronal medium
volume/well

Cell
density/well

Total number
of cells required Plate

6-well 8 cm 2 2.5 ml 2 × 10 6 ≅ 12 × 10 6

24-well 1.9 cm 2 600 μ l 0.38 × 10 6 ≅ 10 × 10 6

allowed to settle for 3 min and about 2 ml is then added to the
second tube (at this stage, there is approximately 11 ml of dissoci-
ated cell suspension). The dissociated cells are centrifuged at 200 g
(10 min, at room temperature). The supernatant is discarded and
the pellet is resuspended in 5 ml of HBSSm by uptake into and
release from fi re-polished Pasteur pipettes. The resuspended pellet
is allowed to settle on ice for 3 min and about 4 ml is transferred
to a new tube. We added 5 ml of HBSSm and centrifuged again for
10 min at 200 g (room temperature). The supernatant is discarded
and the pellet is resuspended in 5 ml of neuronal medium culture
(see Sect. 2 for composition) with a 5-ml pipette. Cell density is
determined and the cells are plated to give a density of 2 × 10 5 cells/
cm 2 (Table 3 ). Striatal primary cultures (mixture of neuronal and
astroglial cells) can be kept for up to 8 weeks, with the replacement
of half the medium each week.

Striatal cultures are transduced with LV-VSV-G at 1 division
(DIV), with 10 ng of p24 antigen/10 5 cells (i.e., 180-200 ng of
p24 antigen from the virus/well for a 6-well plate and 35-40 ng of
p24 antigen from the virus/well for a 24-well plate). With a GFP
virus, fl uorescence is detected in the soma and processes 7 days
after transduction. Fluorescence is maximal 3 weeks after transduc-
tion. Under these conditions, 90-95 % of neuronal cells are infected
and express reporter genes or genes of interest [ 32 ]. When LVs
expressing wild-type or mutant Htt (Htt171-18Q/82Q, respec-
tively) were used to develop an in vitro model of HD, mHtt
resulted in a slowly progressing disease characterized by the appear-
ance, after 1 month, of neuritic aggregates, followed by intranu-
clear inclusions, neuronal dysfunction and, fi nally, cell death at 6-8
weeks [ 32 ]. Immunohistochemical analysis is carried out on fi xed
cells to detect human Htt aggregates (Fig. 8 ). The primary cul-
tures are washed with cold PBS and fi xed by incubation in 4 %
paraformaldehyde for 10 min at 4 °C. They are then washed with
PBS and incubated in a blocking solution of PBS supplemented
with 10 % normal goat serum and 0.03 % Triton X-100. The cells
are then incubated overnight at 4 °C in blocking solution contain-
ing the 2B4 antibody recognizing the N-terminal part of Htt at a
dilution 1/10 3 and for 2 h with the secondary antibody coupled to
a fl uorophore or biotin ( see Note 23 ).

3.8.2 Transduction
and In Vivo Modeling of HD
in Cocultures of Primary
Striatal Neurons