Viral Vectors to Study Synaptic Function - Viral Vector Approaches in Neurobiology and Brain Diseases - page 111

Here, I describe an example of this type of studies. I detail the
methodology for a sindbis virus-based expression system to infect
hippocampal neurons in vivo. This manipulation is then followed
by electrophysiological analysis of synaptic transmission and plas-
ticity in acute slices. This type of protocol can be adapted for any
type of neurotropic virus in any brain region of interest, thus mak-
ing this method extremely powerful to study the involvement of
specifi c proteins in synaptic function.

2 Materials

2.1 Generation of
Sindbis Virus Particles

In this section, we provide a list of the materials necessary for clon-
ing, production, and in vivo injection of sindbis viruses as well as
for electrophysiological recording of synaptic function. Common
reagents and equipment used for general molecular biology or cell
culture manipulations are not listed.

1. Sindbis viral vector backbone pSINRep(nsP2S 726 ) or
pSINRep(nsP2S 726 )-IRES-GFP (Fig. 1a ) [ 20 ]
2. cDNA of protein-of-interest
3. Custom made primers for cloning (see Sect. 3.1.1 and Fig. 2b )
4. PfuI polymerase enzyme and reagents

2.1.1 Cloning into
Sindbis Virus-Based
Vectors

Fig. 1 ( a ) Vector maps of pSINRep(nsP2S 726 ) and pSINRep(nsP2S 726 )-IRES-GFP. The unique restriction sites are
annotated in the multiple cloning site (MCS) and for linearization of the vector prior to virus production. ( b )
Sequencing primer pSINREP5 used to sequence insert