The pGL4.33[luc2P/SRE/Hygro] Vector(a–c) contains a Serum Response Element (SRE) that drives transcription of the luciferase reporter gene luc2P in response to activation of MAPK/ERK signaling pathway. luc2P is a synthetically derived luciferase sequence with humanized codon optimization. The luc2P gene also contains hPEST, a protein destabilization sequence. The protein encoded by luc2P responds more quickly to induction than the protein encoded by the luc2 gene. The vector backbone contains an ampicillin resistance gene to allow selection in E. coli and the mammalian selectable marker for hygromycin resistance.
Sample Protocol to Determine Induction of Luciferase by FBS + PMA in HEK293 Cells Transfected with the pGL4.33[luc2P/SRE/Hygro] Vector
實驗材料
Dulbecco's PBS (DPBS)
0.05% (w/v) trypsin in DPBS
DMEM
DMEM supplemented with 0.5%, 10% and 40% fetal bovine serum (DMEM/FBS)
Phorbol 12-myristate 13-acetate (PMA, Promega Cat.# V1171 or Sigma Cat.# P8139),
1mg/ml solution in DMSO
ONE-Glo Luciferase Assay System (Cat.# E6110)
HEK293 cells
transfection reagent
Day 1: Plate Cells
1. Grow HEK293 cells in DMEM/FBS to approximately 75% confluency.
2. Harvest cells via trypsinization: Remove the DMEM/FBS, wash the cells with DPBS
and add the trypsin/DPBS (1X volume). After 2 minutes, add a 4X volume of
DMEM/FBS, collect the cell suspension and pellet the cells by centrifugation.
Aspirate the supernatant, and resuspend in DMEM/FBS. We have routinely used a
concentration of 10,000–15,000 viable cells/100μl DMEM/FBS.
3. Dispense 100μl of the cell suspension into the wells of a 96-well plate. Plate enough
wells to perform each test condition in triplicate.
4. Cover the plate, and place it in a tissue culture incubator at 37°C overnight (or for 24 hours).
Day 2: Transfect Cells
1. Transfect the cells using a high-efficiency transfection reagent. Each well of cells in a 96-well plate requires 0.1μg pGL4.33[luc2P/SRE/Hygro] Vector DNA. Transfection conditions may require optimization.
2. Cover the plate, and place it in a tissue culture incubator at 37°C.
3. After 4–6 hours, change the medium to DMEM/0.5%FBS (100μl per well) to start
serum starvation.
Day 3: Induce Transfected Cells
1. Prepare 2X induction and 2X control solutions. Calculate the volume of 2X induction
and 2X control solution by multiplying the number of wells needed for each solution
by 50μl, and prepare 110% of this amount.
2X induction solution: 40%FBS plus 20ng/ml PMA in DMEM
2X control solution: DMEM
2. Remove 50μl of medium from wells that will be treated with either 2X induction solution or 2X control solution.
3. Add 50μl of 2X induction solution to the cells to be induced and 50μl of 2X control
solution to the control noninduced cells.
4. Return the plate to the tissue culture incubator, and induce for 6 hours.
5. Analyze luciferase activity using an appropriate luciferase detection assay. We have
observed comparable results for fold induction of the vector using a variety of
luciferase reagents, including: Bright-Glo Luciferase Assay System (Cat.# E2610,
Technical Manual #TM052); ONE-Glo Luciferase Assay System (Cat.# E6110,
Technical Manual #TM292); Dual-Luciferase Reporter Assay System (Cat.# E1910,
Technical Manual #TM040); and Dual-Glo Luciferase Assay System (Cat.# E2920,
Technical Manual #TM058).
6. Calculate the fold induction as follows:
fold induction = average relative light units of induced cells
average relative light units of control cells
