pGL4.43載體簡介
The pGL4.43[luc2P/XRE/Hygro] Vector contains three copies of a xenobiotic response element (XRE) 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 pGL4.43[luc2P/XRE/Hygro] Vector is used to measure
activation of the XRE in HepG2 cells upon treatment with TCDD. 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)
TCDD (2,3,7,8-Tetrachlorodibenzo-p-dioxin; AccuStandard Cat.# D-404N)
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 pGL4.43[luc2P/XRE/hygro] and pGL4.75 [hRluc/CMV] Renilla luciferase
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 4.5:1 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 18 hours
in a 37°C, 5% CO2 incubator.
Day 3: Medium Replacement and Cell Treatment
1. Resuspend TCDD to 31.6μM in DMSO. Serially dilute by half logs into DMSO to
give concentrated stock solutions (316X). Dilute these 31.6-fold into Opti-MEM I
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 TCDD dilutions and incubate for 24 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).
