Viral Vectors to Study Synaptic Function - Viral Vector Approaches in Neurobiology and Brain Diseases - page 116

A new generation of viral backbone vectors was designed to
reduce this toxicity [ 20 ]. The low-toxicity vector pSINRep(nsP2S 726 )
(Fig 1a ) contains a point mutation in the second nonstructural
protein (nsP2), which delays the inhibition of host protein synthe-
sis. Kim et al. 2004 [ 20 ] also constructed an optimized helper vec-
tor [DH-BB(tRNA/TE12)] to use with this new backbone vector
for production of particles with low levels of helper RNA packag-
ing and high neurospecifi city of infection.
For identifi cation of infected cells for electrophysiological
recordings, we have generally used the green fl uorescent protein
(GFP) and have therefore constructed the pSINRep(nsP2S 726 )-
IRES-GFP vector (Fig. 1a ) [ 24 ]. The internal ribosomal entry site
(IRES) allows for coexpression of the protein of interest and GFP
in the same cells without the need for fusion proteins. This how-
ever limits the maximum size of the cDNA encoding the protein-
of-interest to little more than 2.5 kb.
In this section, I provide procedures for cloning, production,
and in vivo injection of sindbis viruses in the rat hippocampus. I
also provide guidelines for electrophysiological analysis of synaptic
function. I have however not detailed standard molecular and cel-
lular biology techniques (e.g., digest with restriction enzymes and
splitting of cells in culture).

Here, I review the cloning procedure to engineer recombinant
sindbis virus vectors. Since each cloning will be unique and
designed to fi t the compatibility of the insert with the vector, the
specifi c cloning strategy should be adapted in each case. As a guide,
I describe the cloning strategy that we generally use to insert the
cDNA of proteins of interest into the sindbis virus vector contain-
ing IRES-GFP (Fig. 2a ). As this method relies on PCR amplifi ca-
tion of the insert with primers that contain suitable restriction sites
for cloning into either pSINRep(nsP2S 726 ) or pSINRep(nsP2S 726 )-
IRES-GFP, this procedure should in principle be adaptable for
cloning most inserts into these vectors. It is necessary that the
insert to be cloned does not contain restriction sites used during
the cloning or linearization steps. This cloning strategy can be
adapted for other types of cloning using the multiple cloning sites
available in the sindbis vectors (Fig. 1a ). I do not detail each step
in this cloning (e.g., PCR amplifi cation, restriction digests, etc.),
but specifi cs can be found in standard molecular biology manuals
and kit manuals.
Primers are designed to amplify the 5

3.1.1 Cloning into
Sindbis Virus-Based
Vectors

ends of the insert
cDNA. A diagram of these primers is depicted in Fig. 2b . The 5

′ and 3 ′

end primer, called here P1, should also contain an XbaI restriction
site and a Kozak sequence to optimize initiation of translation of
your insert. The 3

′

end primer, called here P2, should contain a
stop and a SphI restriction site. I recommend use of the XbaI and
SphI enzymes for cloning in the sindbis vectors as we have proven

′