The pGL4.41[luc2P/HSE/Hygro] Vector contains four copies of a heat shock response element (HSE) 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 pGL4.41[luc2P/HSE/Hygro] Vector is used to measure activation of the HSE in HepG2 cells upon treatment with 17-AAG or CdCl2. 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
DMEM (Life Technologies Cat.# 11995)
Complete medium [DMEM supplemented with 10% fetal bovine serum (DMEM/FBS;
Life Technologies Cat.# 16000] and 1X NEAA [Life Technologies Cat.# 11140])
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)
17-AAG (17-(Allylamino)-17-demethoxygeldanamycin; Calbiochem Cat.# 100068)
CdCl2 (Sigma Cat.# 202908)
DMSO (Sigma Cat.# D2650)
Dual-Glo Luciferase Assay System (Cat.# E2940)
HepG2 cells
pGL4.75[hRluc/CMV] Vector (Cat.# E6931)
實驗流程
Day 1: Plate Cells
1. Grow HepG2 cells in complete medium (DMEM + 10% FBS + 1X NEAA). Wash
twice with DPBS and treat with one volume of 0.05% trypsin-EDTA, followed by four
volumes of complete medium.
2. Vigorously resuspend the cells by pipetting and allow cell clumps to settle. Remove
the cell suspension from any cell clumps, quantify the cells and dilute in complete
medium to 1 × 105 cells/ml.
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 pGL4.41[luc2P/HSE/hygro] and pGL4.75 [hRluc/CMV] Renilla luciferase
vector constructs in a 10:1 mass ratio, respectively, to 10ng total DNA/μl in
Opti-MEM I.
2. Add FuGENE HD to a 4.5:1 lipid:DNA ratio. Mix by pipetting. Incubate at room
temperature for 20 minutes.
3. Add 10μl transfection complex per well (100ng DNA/well) and incubate for 18 hours
in a 37°C, 5% CO2 incubator.
Day 3: Medium Replacement and Cell Treatment
1. Resuspend 17-AAG (17-(Allylamino)-17-demethoxygeldanamycin) to 1mM in
DMSO. Serially dilute into DMSO to give concentrated stock solutions (1,000X).
Serially dilute a 1mM aqueous stock of CdCl2 into water to give concentrated stock
solutions (1,000X). Dilute the 1,000X stocks of 17-AAG and CdCl2 into DMEM to
give 10X stocks.
2. Remove existing medium from cells and replace with 72μl of DMEM + 0.5%
charcoal-stripped FBS per well.
3. Add 8μl of the 10X dilutions of 17-AAG or CdCl2 and incubate for 6 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).
