The pGL4.38[luc2P/p53 RE/Hygro] Vector contains two copies of a p53 response element (p53 RE) that drives transcription of the luciferase reporter gene luc2P (Photinus pyralis). luc2P is a synthetically-derived luciferase sequence with humanized codon optimization that is designed for high expression and reduced anomalous transcription. The luc2P gene contains hPEST, a protein destabilization sequence, which allows luc2P protein levels to respond more quickly than those of luc2 to induction of transcription. The vector backbone contains an ampicillin resistance gene to allow selection in E. coli and a gene for hygromycin resistance to allow selection of stably transfected mammalian cell lines.
Example Protocol
In this example protocol, the pGL4.38[luc2P/p53 RE/Hygro] Vector is used to measure
activation of the p53 RE in U2OS cells upon treatment with doxorubicin, etoposide, nutlin-
3, or mitomycin c. The pGL4.75 Vector (encoding Renilla luciferase) is used as a
normalization control. In designing such experiments, it is important that the chosen cell
type can be transfected efficiently and that it expresses the proper components of the signaling
pathway of interest in order to generate the biological response. Protocol optimization
may be required for your particular cell type and assay conditions.
實驗材料
Complete medium [McCoy’s 5A (Life Technologies Cat.# 16600) + 10% FBS (Life
Technologies Cat.# 16000) + 1X NEAA (Life Technologies Cat.# 11140) + 1X
sodium pyruvate (Life Technologies Cat.# 11360)]
Dulbecco’s PBS (DPBS; Life Technologies Cat. # 14190)
0.05% Tryspin-EDTA (Life Technologies Cat.# 25300)
Charcoal-stripped FBS (Life Technologies Cat.# 126776-011)
Opti-MEM I (Life Technologies Cat.# 31985)
FuGENE HD Transfection Reagent (Cat.# E2311)
Doxorubicin (Sigma Cat.# D1515)
Etoposide (Calbiochem Cat.# 341205)
Mitomycin c (Sigma Cat.# 705436)
Nutlin-3 (Sigma Cat.# N6287)
DMSO (Sigma Cat.# D2650)
Dual-Glo Luciferase Assay System (Cat.# E2940)
U2OS cells
pGL4.75[hRluc/CMV] Vector (Cat.# E6931)
實驗流程
Day 1: Plate Cells
1. Grow U2OS cells in complete medium (McCoy’s 5A + 10% FBS + 1X NEAA + 1X
sodium pyruvate). Wash with DPBS and treat with one volume of 0.05% trypsin-
EDTA. Resuspend the cells in four volumes of complete medium.
2. Quantify the cells and dilute to 1 × 105 cells/ml in complete medium.
3. Plate 100μl per well to a solid, white 96-well plate (Corning Cat.# 3917).
4. Incubate for 24 hours in a 37°C, 5% CO2 incubator.
Day 2: Transfection
1. Dilute pGL4.38[luc2P/p53 RE/Hygro] and pGL4.75 [hRluc/CMV] Renilla luciferase
control vector constructs in a 10:1 mass ratio, respectively, to 12.5ng total DNA/μl in
Opti-MEM I.
2. Add FuGENE HD to a 3:1 lipid:DNA ratio. Mix by pipetting. Incubate at room temperature
for 20 minutes.
3. Add 8μl transfection complex per well (100ng DNA/well) and incubate for 24 hours
in a 37°C, 5% CO2 incubator.
Day 3: Medium Replacement and Cell Treatment
1. Resuspend doxorubicin to 50mM in water. Resuspend etoposide to 50mM in DMSO.
Resuspend mitomycin c to 1mM in water. Resuspend nutlin-3 to 10mM in DMSO.
Make serial dilutions in either water or DMSO and then dilute into Opti-MEM I to
make 10X stocks.
2. Remove existing medium from cells and replace with 72μl of McCoy’s 5A + 0.5%
charcoal-stripped FBS per well.
3. Add 8μl of the 10X compound dilutions and incubate for 18 or 40 hours in a 37°C,
5% CO2 incubator.
Day 4: Luminescence Measurement
1. Remove plates from the 37°C, 5% CO2 incubator and allow to cool to room temperature
for approximately 15 minutes.
2. Add 80μl of the Dual-Glo Luciferase Assay System detection reagents and measure
luminescence following the recommended protocol (Refer to the Dual-Glo
Luciferase Assay System Technical Manual, #TM058 for details).
