The pGL4.39[luc2P/ATF6 RE/Hygro] Vector contains five copies of an ATF6 response element (ATF6 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 pGL4.39[luc2P/ATF6 RE/Hygro] Vector is used to measure
activation of the ATF6 RE in HeLa cells upon treatment with tunicamycin.. 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)
Tunicamycin (Calbiochem Cat.# 654380)
DMSO (Sigma Cat.# D2650)
Dual-Glo Luciferase Assay System (Cat.# E2940)
HeLa cells
pGL4.75[hRluc/CMV] Vector (Cat.# E6931)
實驗流程
Day 1: Reverse Transfection
Preparation of Cells
1. Grow HeLa cells in complete medium (DMEM + 10% FBS + 1X NEAA). Wash with
DPBS and treat with one volume of 0.05% trypsin-EDTA. Resuspend cells in four
volumes of complete medium.
2. Pellet the cells by centrifugation at 200 × g for 5 minutes in a swinging-bucket rotor.
Resuspend in complete medium at a concentration of 1 × 105 cells/ml.
Preparation of Lipid:DNA Mixture
1. Dilute pGL4.39[luc2P/ATF6 RE/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 3:1 lipid:DNA ratio. Mix by pipetting. Incubate at room temperature
for 30 minutes.
3. Dilute lipid:DNA mixture 20-fold with 1 × 105 cells/ml cell suspension and mix by
inversion.
4. Plate 100μl per well into a solid, white 96-well plate (Corning Cat.# 3917).
5. Incubate for 18 hours in a 37°C, 5% CO2 incubator.
Day 2: Medium Replacement and Cell Treatment
1. Resuspend tunicamycin to 10mM in DMSO. Serially dilute into DMSO to give
1,000X stocks. Dilute 100-fold 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 tunicamycin and incubate for 18 hours in a 37°C,
5% CO2 incubator.
Day 3: Luminescence Measurement
1. Remove plates from the 37°C, 5% CO2 incubator and allow to cool to room temperature
for approximately 15 minutes.
4. 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).
