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 228

(P = 0.008) which were both significant relative to transfected untreated cells or
cells stimulated with either drug alone (n = 3). Although T-cell activation resulted
in an average 2.3 ± 0.2 - fold stronger induction capacity of luciferase reporter ex-
pression from the NFAT3 promoter compared to the IL-2 promoter (P < 0.001),
the basal transcriptional activity of the NFAT3 promoter in the absence of T-cell
activation was 4.6 ± 0.2 - fold higher than that of the IL-2 promoter (P < 0.001).
These results indicated that the IL-2 promoter serves as more stringent T-cell
activation-dependent promoter than the synthetic NFAT3 promoter.
Figure 1. Activation-induced luciferase reporter expression in Jurkat TAg cells. A. Jurkat cells that express
the large T Ag were transiently transfected by electroporation with an IL-2 promoter-luciferase construct. Co-
stimulation of these T-cells with 1 µ M ionomycin and 10 ng/ml PMA for ~6 hr resulted in 6.4 ± 0.4 - fold
increase in IL-2 promoter-driven luciferase reporter expression relative to control non-stimulated cells (P =
0.002). B. Similar co-stimulation of Jurkat T Ag cells that were transfected with NFAT3 promoter-luciferase
construct reulsted 14.6 ± 1.3 - fold increase in NFAT3 promoter-driven luciferase expression relative to control
(P = 0.008).
synthesis of the siniL-2pr retrovector
To develop a retroviral vector for activation-induced stable transgene expression
in T-cells, we generated a self-inactivating retroviral vector (SIN) by creating ex-
tensive deletions to the U3 region of the Moloney Murine Leukemia Virus 3' long
terminal repeat (3'LTR) in pGFP/TKfus (Figure 2A). Retroviral promoter and
enhancer machinery were replaced by the full-length human IL-2 promoter as de-
scribed in the methods section. The resulting construct SINIL-2pr incorporated
the EGFP fused to HSVTK as a reporter/suicide gene (Figure 2B). Subsequently,
the SINIL-2pr plasmid was stably transfected into the 293GPG packaging cell