Alphavirus-Based Vectors - Viral Vector Approaches in Neurobiology and Brain Diseases - page 94

were removed from 3- to 5-day-old rats, and the neurons recovered
by trypsin (10 mg/mL type XI, 0.5 mg/mL DNase I type IV) and
mechanical dissociation. The cells were cultured in minimal essen-
tial medium containing 0.6 % (wt/vol) glucose, 1 mM glutamine,
2.4 g/L NaHCO3, 100 mg/mL bovine transferrin, 25 mg/mL
insulin, and 5-10 % FCS. The cells were plated at a density of
50,000 cells per 35 mm plastic Petri dish (Falcon) coated with
poly-ornithine and Matrigel (Collaborative Research). The cul-
tures were maintained at 37 °C in 95 % air, 5 % CO 2 in a humidi-
fi ed incubator, and the medium replaced every 3-4 days. From the
second day in culture, the culture medium was supplemented with
5 M cytosine-Darabinofuranoside. Neurons were used after 10-14
days in culture.
Primary hippocampal neurons were isolated from embryos
(stage E17) of rat strain ROROspf120 (BRL, Fullinsdorf,
Switzerland) and cultured in D-MEM (Gibco BRL) supplemented
with 10 % horse serum. Organotypic slice cultures from rat hip-
pocampus were prepared in the roller-tube confi guration as previ-
ously described [ 23 ].

2.2 SFV Plasmid
Vectors

Replication-defi cient recombinant particles were generated from
expression vectors pSFV1 and pSFV2gen (also called pSFV4.2)
together with the pSFV-Helper2 vector (Fig. 2 ). Less cytotoxic
and temperature-sensitive vectors based on pSFV2gen have been
engineered [ 16 , 17 ]. The vectors pSFV1 and pSFV-Helper2 were
linearized by Spe I and pSFV2gen and its derivatives by Nru I.

1. Restriction endonucleases Spe I, Nru I (Roche Molecular
Biochemicals)
2. 0.8 % Agarose gel (Q-Biogene)
3. Gel electrophoresis apparatus (BioRad)
4. Phenol/chloroform/isoamyl alcohol 25:24:1 (v/v/v) (Gibco
BRL)
5. 3 M Sodium acetate, pH 4.8 (Fluka)
6. 95 % and 70 % (v/v) Ethanol (Merck)
7. 10× SP6 Buffer (400 mM HEPES, pH 7.4, 60 mM magne-
sium acetate, 20 mM spermidine)
8. 10 mM m7G(5

2.3 Reagents
and Equipment

′ )ppp(5 ′ )G (sodium salt: Roche Molecular

Biochemicals)
9. 50 mM Dithiothreitol (DTT) (Fluka)
10. rNTP Mix (10 mM rATP, 10 mM rCTP, 10 mM rUTP, and
5 mM rGTP) (Roche Molecular Biochemicals)
11. 10-50 U/

μ L RNase inhibitor (Roche Molecular Biochemicals)

12. 10-20 U/ μ L SP6 RNA polymerase (Amersham Pharmacia

Biotech)