Alphavirus-Based Vectors - Viral Vector Approaches in Neurobiology and Brain Diseases - page 93

and Sindbis virus particles into rat organotypic hippocampal slice
cultures has allowed gene expression and functional studies [ 15 ].
The injection of SFV and Sindbis vectors resulted in highly specifi c
expression of GFP in neuronal cells. It was estimated that more
than 90 % of the GFP-positive cells were of neuronal origin. The
infected neurons remained viable for 5 days postinfection deter-
mined by propidium iodide exclusion, which is suffi cient for
electrophysiological recordings. Furthermore, application of
temperature-sensitive SFV vectors demonstrated a strong prefer-
ence for expression in interneurons and not in pyramidal cells as is
the case with the wild-type vector [ 16 ]. Moreover, the less cyto-
toxic SFV-PD showed enhanced expression levels and prolonged
expression times [ 17 ]. An SFV vector based on the avirulent
A7(74) strain generated temperature-dependent expression in slice
cultures [ 18 ]. At 37 °C, the majority of the GFP-positive cells were
glial cells, whereas at 31 °C the GFP expression was located mainly
in neurons. Extensive studies on the localization and functional
activity of Homer/Vesl proteins, regulators of metabotropic gluta-
mate receptors, have been conducted with SFV vectors in hippo-
campal slice cultures [ 19 ]. Alphavirus vectors have also been
applied for in vivo administration in rodent brain [ 20 , 21 ]. Sindbis
virus was used for successful high level delivery and -galactosidase

expression in mouse nucleus caudate/putamen and nucleus accum-
bens septi [ 20 ]. Similarly, SFV-LacZ was injected into the amyg-
dale and striatum of male Wistar rats [ 21 ]. Local transient
β

β

-galactosidase expression was observed at the injection sites with-
out any spread into other brain regions. The injected animals
showed no signifi cant difference in body weight and temperature,
exploratory behavior and forced motor performances in comparison
to control animals.

2 Materials

BHK-21 (baby hamster kidney) cells were used for in vivo packaging
of recombinant SFV particles. They were cultured in a 1:1 mixture
of Dulbecco's modifi ed F-12 medium (Gibco BRL) and Iscove's
modifi ed Dulbecco's medium (Gibco BRL) supplemented with
4 mM glutamine and 10 % fetal calf serum (FCS). CHO-K1
(Chinese hamster ovary) and HEK293 (human embryonic kidney)
cells used for expression studies were grown in the same medium.
Application of alternative cell lines may require special media.
Primary cultures of dispersed neurons were obtained from
embryonic day 18 (E18) rat hippocampal and cortical neurons and
cultured in Neurobasal medium (Invitrogen) on 24-well plates,
while postnatal day 4-5 (P4-5) rat hippocampal neurons were
cultured on glass coverslips in 35 mm Petri dishes as previously
described [ 22 ]. Briefl y, the CA1 and CA3 hippocampal regions

2.1 Cell Cultures