Viral Vectors to Study Synaptic Function - Viral Vector Approaches in Neurobiology and Brain Diseases - page 120

(b) Transfer supernatant from fl ask (12 mL total) to a sterile
15-mL tube and cap tube and centrifuge at 3,000 rpm for
5 min to remove debris.
(c) Transfer supernatant in Beckman ultra-clear centrifuge tube
and centrifuge at 30,000 rpm for 1 h and 30 min at 4 °C.
(d) After end of centrifugation, pick up the Beckman tube care-
fully and remove as much supernatant as possible (try to leave
~200 L) without touching the bottom of the tube. The virus

particles are concentrated at the bottom of the tube, but the
pellet is clear and cannot be detected by eye.
(e) Gently resuspend the invisible virus pellet in the 200

μ

μ L left-

over medium with a 1-mL pipette.
(f) Aliquot 10 μ L of virus into small tubes and store in a −80 °C

freezer.

I have optimized an approximate but rapid titration procedure
to qualitatively evaluate if the viral solution obtained is suitable for
in vivo infection. Here is a brief description of this procedure:

1. Plate 100,000 BHK cells/per well in 6 wells of a 24-well plate
(in 1 mL of aMEM + FBS) in the morning. A coverslip may be
added to each well for more accurate evaluation of infection
percentage using a confocal microscope after fi xation.
2. After 7 h, infect each well with either 5-, 2.5-, 1-, 0.5-, 0.1-,
or 0.01 L of the viral solution aliquoted in step (f).

3. After 24 h incubation at 37 °C, check for percentage of infec-
tion in each well on the live cell using GFP fl uorescence (or
after fi xation and mounting if a coverslip was present in the
wells). We have consistently observed that a viral solution that
displays strong infection (at least 80 %) even in the 0.1

μ

μ L

infected well (sometimes also in the 0.01 μ L well) should pro-

vide good in vivo infection.

We have optimized an in vivo surgery procedure to inject the brain
of rats or mice with recombinant sindbis viruses. This procedure is
described here for infection of CA1 pyramidal neurons of young
adult rats (postnatal days 20-22) but can be adapted to any area
of the brain, at any age as well as for infection of mice (see for
example [ 24 ]).
(a) Inject rat with an anesthetic by intraperitoneal (IP) injection.
We recommend use of a cocktail of ketamine/xylazine. For
juvenile rats, the cocktail consists of 2.4 mL ketamine
(100 mg/mL), 1.1 mL xylazine (20 mg/mL), and 6.5 mL
sterile saline. We inject it at a dose of 0.1 mL/30 g of body
weight. Anesthesia parameters should be adapted to the age
of the animal and the species. For mice, we have also used
chloral hydrate as an anesthetic (400 mg/kg) [ 24 ].

3.2 In Vivo
Stereotaxic Injection
of Viruses