Viral Vectors to Study Synaptic Function - Viral Vector Approaches in Neurobiology and Brain Diseases - page 115

9. Vibrotome for slice preparation (e.g., Leica VT series or
Thermo Scientifi c Microm HM650V)
10. Glass electrode puller (e.g., PC-10 from Narishige or P-97
from Sutter Instruments)

All experiments must be conducted in accordance with national
and institutional guidelines for care and use of laboratory animals.

3 Methods

3.1 Generation of
Sindbis Virus Particles

We have chosen the sindbis virus system because it produces the
recombinant protein of interest rapidly (within 24 h) and is highly
neurotropic [ 21 ]. The sindbis virus is a member of the alphavirus
family, like the Semliki forest virus. These viruses are small-
enveloped viruses with single-stranded RNA genomes [ 22 ]. The
sindbis expression system is a transient expression system in which
the sindbis life cycle is exploited to produce recombinant proteins.
The fi rst generation of vectors using the sindbis virus backbone
(pSINRep-5), in which the insert of interest is cloned, also con-
tains the nonstructural genes but lacks the structural viral proteins
normally necessary to package the RNA into viral particles. These
DNA constructs are subsequently used to make genome-length
RNA transcripts in vitro (recombinant RNA). Production of
replication-defi cient infectious viruses is accomplished by trans-
fecting cells with the recombinant capped RNA and a helper RNA
that provides the structural proteins in trans . Particles released by
the transfected cells contain only the recombinant RNA and are
ready to infect new cells for expression studies. These recombinant
viruses will undergo only one round of infection as they do not
contain the helper RNA, which encodes the structural proteins.
Expression of the transgene is detected within a day both in vitro
and in vivo. More information on the sindbis viral system can be
found in [ 21 ] and [ 22 ].
The main advantages of this type of virus in neuroscience are
its high neurotropism (preferentially targeting glutamatergic neu-
rons), the strength and speed of transgene expression, and the
good diffusion of viral particles in vivo. On the other hand, these
viruses lead to cytotoxicity within a few days after infection and are
therefore not suitable for long-term expression studies. This is due
to the fact that the recombinant RNA, once transfected into cells,
promptly recruits the host translational machinery for its own use,
resulting in high levels of the desired protein, but at the expense of
cell viability (it progressively shuts off gene expression in the host
cell). Also, transgene size is limited as packaging effi ciency dimin-
ishes if the insert size is more than 4 kb. In vivo investigations
using this virus have used a time frame of expression of up to 3 days
with success [ 23 ].