Lentiviral Vectors for Gene Delivery to the Nervous System - Gene Delivery and Therapy for Neurological Disorders - page 56

5. At the end of transduction, remove the medium containing
the lentiviral vector and replace with fresh complete growth
medium.
6. Incubate for 72 h, when transgene expression is optimal.
7. For vectors expressing fl uorescent transgenes, expression can
be evaluated 72 h post transduction with a fl uorescence
microscope.
8. For fl ow cytometry analysis of the expression of LV-mediated
gene transfer, dissociate and fi x cells as described in Sect. 3.3.1
steps 7-17.

For cells that are in suspension transduction can be performed
with spinoculation. In this case, a suitable amount (transduction
mix of 250 l, for 24-well plates) of LV is added to cells and plates

are spin at 2,500 rpm for 90 min at 37 °C. Upon spinoculation
plates are transferred to an incubator and incubated for 2 h. Upon
incubation transduction medium is discarded and fresh growth
medium is added to cells ( see Notes 20 and 21 ).

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3.5 In Vivo
Application
of Lentiviral Vectors

In our lab upon lentiviral vector production, titration and extensive
assessment in various immortalized and primary cell lines in vitro,
we proceed to in vivo applications in CNS to examine the neurot-
ropism of our lentiviral preparations and/or to target specifi c
regions with the fi nal aim of developing a potential therapy for
neurological disorders.
Here we present two of the most common in vivo techniques
we are performing.

In all our in vivo applications we use male Wistar rats, weighing
200-250 g.
1. Prior to surgery, all animals are deeply anesthetized by inhala-
tion of a mixture of 1 l/min oxygen and 3.5 % isofl urane
(Merial, Australia) and then receive systemic analgesia.
2. Rats are placed in a stereotactic frame (Taxic-6000, World
Precision Instruments, USA) with the nose bar set at −3.3 mm.

3.5.1 Intrastriatal
Delivery of Lentiviral
Vectors

Procedure

3. The anesthetic mixture is changed to 1 l/min oxygen and
2.2 % isofl urane for the remaining of the operation.
4. The scalp is cut and retracted to expose the skull. Craniotomy
is performed by drilling directly above the target region, to
expose the pial surface. One single injection is directed into
the right striatum using the stereotactic coordinates relative to
bregma: anteroposterior, 0.5 mm; mediolateral, 3.0 mm; dor-
soventral, 5.0 mm.
5. Animals usually receive 4.0

l of pseudotyped lentiviral vectors
with a biological titer of 1 × 10 9 TU/ml via a 32 G needle
using an infusion pump (UltramicropumpIII and Micro4

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