A Self-Inactivating Retrovector Incorporating the IL-2 Promoter for Activation- Induced Transgene Expression in Genetically Engineered T-Cells - Genetic Engineering: Recent Developments in Applications - page 237

AC T CTT GAA CAA GAG antisense primer (Sheldon Biotechnology Centre,
McGill University, Montreal). The amplified insert was then digested with the
corresponding enzymes, purified, and ligated into the SIN-derivative in order to
generate the SINIL-2pr plasmid (Figure 2). Nucleotide sequences of the mutated
3'LTR and the inserted IL-2 promoter were confirmed by DNA sequencing (Ge-
nAlyTic Inc., University of Guelph, Ontario).
Generation of the siniL-2pr retroviral Producers and
Production of VSV-G Pseudotyped Retroviral Particles
The SINIL-2pr retroviral producers were generated by stable transfection of the
293GPG packaging cell line as previously described [35]. In brief, stable producer
cells were generated by co-transfection of 5 µ g FspI-linearized SINIL-2pr ret-
rovector and pJ6 Ω Bleo plasmid at a 10:1 ratio. Transfected packaging cells were
subsequently selected in 293GPG media supplemented with 100 µ g/ml Zeocin
(Invitrogen, San Diego, CA) for 3-4 weeks. Resulting stable polyclonal as well
as isolated single clone producer populations were utilized to generate VSV-G
pseudotyped retroviral particles. All viral supernatants were filtered through 0.45-
µ m pore size syringe-mounted filters (Gelman Sciences, Ann Arbor, MI) and
stored at -20°C. Subsequent 100 × concentration of the retroviral preparations
was performed using ultracentrifugation according to a previously described pro-
cedure [35] after which samples were stored at -80°C.
SINIL-2pr Viral Titer Determination and RCR Assay
Jurkat T-cells were suspension-cultured in 12-well plates at ~2 × 10 5 cells per
well per 1 ml of the concentrated retroviral sample serially diluted in complete
RPMI media supplemented with 6 µ g/ml Polybrene (Sigma). Cells transduced
with the various viral dilutions were then incubated overnight at 37°C and 5 %
CO 2 . Afterwards, the samples were spun at 1000 g for 5 min to pellet the cells
and the virus-containing supernatent was discarded. The cells for each sample
were re-suspended in 2 ml fresh complete RPMI media and expanded in culture.
FACS analysis was performed on these samples on 5th day post transduction to
ascertain retrovector expression and gene transfer efficiency as measured by EGFP
fluorescence. Based on the number of target cells per well per dilution sample at
the time of viral addition and assuming that each cell takes 1 retroparticle when
the gene transfer is not saturated (less than 40%), the viral titer was estimated as
~1 × 10 7 infectious particles per ml. Viral preparations were devoid of replication
competent retrovirus (RCR) as determined by standard EGFP marker rescue as-
say performed on null untransduced Jurkat cells exposed to conditioned superna-
tant collected from transduced Jurkat cells.