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Transduction Of Jurkat and P116 T-Cell Lines, Southern Blot,
and drug stimulation
Each of Jurkat and P116 T-cell lines was suspension-cultured at ~2 × 10
5
cells per
well per 1 ml of complete RPMI media. The cells were transduced with SINIL-2-
pr concentrated retrovirus at an MOI of 5 in whole media supplemented with 6
µ
g/ml Polybrene. This procedure was repeated daily for ~6 hr exposure for three
consecutive days after which stably transduced cells were expanded. No clonal
selection was performed, and mixed populations of transduced cells were used for
all subsequent experiments. Southern blot analysis was performed on 15
µ
g of
overnight KpnI digested genomic DNA extracted from stably transduced cells as
well as untransduced control cells. Blots were hybridized with P32 labeled cDNA
probes, washed and exposed on photographic film. T-cell stimulation was done
using 1
µ
M ionomycin and/or 10 ng/ml PMA in whole media for ~6 hr and in
some experiments following 30 min exposure to 300 nM cyclosporin A (CsA)
(Novartis Pharma Canada Inc.). FACS analysis was performed to ascertain ret-
rovector expression as measured by mean EGFP fluorescence of positively gated
cells.
statistics
Student T test was applied using Microsoft Excel software.
competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
DJ carried out the cloning and generation of the various constructs, retroviral
production and titer assays, transduction and subsequent analysis of target cells,
activation assays, luciferase assays, data acquisition and analysis, and drafted the
manuscript. LL did the Southern Blot, and revised the manuscript. CC contrib-
uted to the conception of the designs in the study, acquisition of funding, and
revised the manuscript. JG had substantial contribution to the conception of the
study, the experimental designs, general supervision of the research, acquisition of
funding, and critically revised the manuscript. All authors approved of the final
manuscript version.
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