Alphavirus-Based Vectors - Viral Vector Approaches in Neurobiology and Brain Diseases - page 105

necessary, it is recommended that virus stocks are fi lter-sterilized
(see Sect. 3.6 ) to ensure that no cell debris or contaminants are
injected ( see Note 5 ). Male Wistar rats (lbm RoRo, SPF, Biological
Research Labs Ltd., Switzerland) have been used [ 21 ]. The rats
were stereotactically microinjected under general anesthesia with
Ketamine/Xylazine (200/10 mg/kg ip) in physiological saline
under thermoregulatory control and oxygen supplementation.
Craniotomy was conducted with a fi ne dental drill and injections
performed according to coordinates described earlier [ 25 ]. Attach
stainless steel injectors to a stereotaxic holder, and lower the needle
into the target area. Use appropriate amount of virus (10 5 particles
into striatum/amygdala/cortex and 10 7 particles into ventricles)
using stainless steel injectors. Apply a microinfusion pump (Harvard
PHD 2000) to transfer the virus solution via polyethylene tubing
to a 10- μ L Hamilton syringe. Infuse solutions in a volume of 1 μ L

over 2 min (0.5 L/min). Leave the injection needle in place for

an additional 2 min before slow withdrawal over 1 min. Suture the
wound and keep animals warm for 3-4 h postsurgery. If required,
SFV stocks can be concentrated and the medium replaced by any
method described in Sects. 3.11.1 , 3.11.2 , and 3.11.3 . However,
our in vivo study in rat striatum and amygdala did not reveal any
inconvenience of using SFV in the original culture medium for
BHK cells. In the case of virus concentration and replacement of
medium, it is advisable to make a new titration after the procedure.
It is worth mentioning that some components can be toxic to SFV.
It was found that CSF (cerebrospinal fl uid) is toxic to SFV and
therefore substantially decreased the titers when virus stocks were
resuspended in CSF (Lundstrom, unpublished results).

μ

4 Notes

1. The quality of the DNA preparations is essential for obtaining
good quality RNA necessary for generating high-titer virus
stocks. It is strongly advisable to use commercial Midiprep of
Maxiprep DNA kits for plasmid DNA preparation.
2. The optimal composition of the in vitro transcription reaction
is very important, especially the concentration of the CAP ana-
logue m 7 G(5 )G. Particularly the use of commercial

buffers should be critically evaluated as although high RNA
yields might be obtained the virus titers might be low.
3. The quality of the in vitro transcribed RNA is crucial for effi -
cient virus production. Furthermore, the length of the insert
introduced into the expression might affect the obtained RNA
yields. For vectors exceeding 4 kb inserts RNA yields might be
improved by extending the incubation time for the in vitro
transcription reaction.

′ )ppp(5 ′