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 236

transfection of Jurkat tAg cells and drug stimulation
20 × 10 6 Jurkat T-cells expressing the large T antigen were co-transfected with a
total of ~10 µ g DNA of either the IL-2 promoter-luciferase or the NFAT3 pro-
moter-luciferase plasmid and PEF-GFP (empty vector) at a ratio of 10:1. A con-
trol sample was transfected with ~10 µ g PEF-GFP DNA only to normalize for the
efficiency of transfection. Transient transfections were performed using standard
electroporation at 960 µ F and 240 V (Bio-Rad, Hercules, CA). The samples were
then cultured in 75-cm2 tissue culture flasks in complete RPMI media at 37°C
and 5%CO2 for two days. Afterwards, samples were analyzed by flow cytometry
(FACStar sorter, Becton Dickinson, Mountain View, CA) to determine transfec-
tion efficiency based on EGFP fluorescence. Each sample was then divided into 4
subsets that were either stimulated with 1 µ M ionomycin (Sigma, St. Louis, MO)
or/and 10 ng/ml PMA (Sigma) or left untreated for ~6 hr.
Luciferase Assays
Transfected Jurkat TAg cells were harvested ~6 hr post stimulation and washed
twice with phosphate-buffered saline (PBS). Afterwards, cells in each sample were
lysed using 250 µ l lysis buffer (Luciferase Reporter Gene Assay, Boehringer Man-
nheim). Then, a 20 µ l aliquot of each lysate supernatant was mixed with 50 µ l
luciferase reagent buffer containing the luciferin substrate (Luciferase Reporter
Gene Assay, Boehringer Mannheim). Within 30 sec of starting the reaction per
sample, luciferase activity was measured as light units based on light emission
at 562 nm using the Lumat LB-9507 luminometer (Perkin Elmer Instruments,
Rodgau-Juegesheim, Germany) according to manufacturer's specifications. All
measurements were done in triplicates.
siniL-2pr retrovector design and synthesis
We used a derivative of pGFP/TKfus [33] to generate the SINIL-2pr design.
pGFP/TKfus contains the cDNA for the enhanced green fluorescent protein
(EGFP) reporter and the herpes simplex virus thymidine kinase (HSVTK) sui-
cide gene as a fusion and incorporates the CMV promoter in the 5'LTR which
drives expression in transfected viral packaging cells. We derived a self-inactivat-
ing (SIN) vector from this plasmid by creating a 341-bp NheI-AscI/Klenow dele-
tion in the 3'LTR to remove all the retroviral promoter and enhancer machinery.
An insert encoding for the human IL-2 promoter (-372 to +48) was generated
by PCR (PTC-100™ Programmable Thermal Controller, MJ Research Inc.) us-
ing CTA GCT AGC (introducing NheI site) GAA AGG AGG AAA AAC TGT
TTC AT sense primer and C GAG CTC (introducing Ecl136II site) TAG GGA