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enhancer region. The 5' half of the env gene (nt 6582 to 7981) was also removed,
leaving the RRE (REV-responsive element) sequence and the 5' and 3' exons of
the tat and rev regulatory genes intact. The 3' LTR (nt 9444 to 10249) was re-
placed by a SV40 polyadenylation sequence, resulting in deletion of the 3' end of
the nef gene. Finally, the nef initiation codon was inactivated to prevent transla-
tion (figure 1B).
pGREV was used for pseudotyping. It is a bicistronic expression construct
encoding the vesicular stomatitis virus glycoprotein (VSV-g) and the REV regula-
tory protein, linked by an EMCV IRES. Expression of this cassette, which con-
tains the rabbit
β
-globin intron II and polyadenylation (pA) sequences (figure
1C), is driven by the constitutive CMV promoter.
Production of SIV Vectors
293T cells were plated at a density of 4.0 × 10
5
cells per well (in 6-well plates) on
the day before transfection. Cells were transfected as previously described[42].
SIV vectors were produced by cotransfection with three plasmids: the SIV plas-
mid vector (pRMES8 or pGASE)(1.7
µ
g), the helper plasmid, pSIV3
+
, encoding
Gag-Pol and regulatory proteins other than Env and Nef (1.7
µ
g) and the enve-
lope glycoprotein-encoding plasmid pGREV (2.2
µ
g). The transfection medium
was replaced after 16 hours of incubation. Virus-containing medium was col-
lected 40 hours after transfection, clarified by centrifugation for 5 minutes at 800
g, and passed through a filter with 0.45
µ
m pores. For high-titer preparations,
SIV vectors were concentrated by ultracentrifugation at 110,000 g for 2 hours.
The viral pellet was resuspended by incubation for 2 hours at 4°C in phosphate-
buffered saline supplemented with 1% glycerol[34].
For determination of the infectious titer, sMAGI cells were seeded at a den-
sity of 4 × 105 cells/ml in six-well plates one day before transduction in DMEM
medium (Life Technologies Inc., Berlin, Germany) supplemented with 10% fetal
bovine serum (FBS) (Gibco BRL, Grand Island, New York, USA), polybrene
(6 µg/ml) (Sigma, Saint Louis, USA) and an antibiotic mixture (5 mg/ml peni-
cillin; 5 mg/ml streptomycin; 10 mg/ml neomycin; Gibco BRL, Grand Island,
New York, USA). The cells were cultured for one day, and we then added serial
dilutions of virus preparations and incubated the plates for a further four hours.
Cells were then washed in DMEM (Life Technologies Inc., Berlin, Germany).
Transduction rates was determined 48 hours after infection, as the percentage
of GFP-positive sMAGI cells (%GFP+c), by flow cytometry (FACScan, Becton
Dickinson, San Jose, Mountain View, California, USA) after transducing 4 × 105
cells with 1 ml of diluted viral supernatant (dilution factor = d). The infectious
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