The pGL4.47[luc2P/SIE/Hygro] Vector contains five copies of the SIS-inducible element (SIE) 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 pGL4.47[luc2P/SIE/Hygro] vector is used to measure
activation of the SIE in HEK293 cells upon treatment with interleukin 6. 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.
实验材料
DMEM (Life Technologies Cat.# 11995)
FBS (HyClone Cat.# SH30070.03)
Dulbecco’s PBS (DPBS; Life Technologies Cat. # 14190)
0.05% Tryspin-EDTA (Life Technologies Cat.# 25300)
Opti-MEM I (Life Technologies Cat.# 31985)
FuGENE HD Transfection Reagent (Cat.# E2311)
Human recombinant interleukin 6 (IL-6, Life Technologies Cat.# PHC0061)
DMSO (Sigma Cat.# D2650)
ONE-Glo Luciferase Assay System (Cat.# E6120)
HEK293 cells
实验流程
Day 1: Plate Cells
1. Plate 10ml of HEK293 cells at 2 × 105 cells/ml in a 10cm dish in complete medium
(DMEM + 10% FBS).
2 Incubate for 24 hours in a 37°C, 5% CO2 incubator.
Day 2: Transfection
1. Dilute 10μg pGL4.47[luc2P/SIE/Hygro] Vector DNA in 500μl Opti-MEM I.
2. Add 30μl FuGENE HD to a 3:1 lipid:DNA ratio and mix. Incubate at room
temperature for 15 minutes.
3. Add DNA-lipid complex to cells and mix gently to ensure even distribution.
4 Incubate for 18 hours in a 37°C, 5% CO2 incubator.
Day 3: Medium Replacement and Cell Treatment
1. Wash cells with DPBS and treat with one volume of 0.05% trypsin-EDTA.
Resuspend cells in four volumes of complete medium.
2. Quantify the cells and dilute to 2 × 105 cells/ml in complete medium.
3. Plate 50μl per well into a solid, white 96-well plate (Corning Cat.# 3917).
4. Serially dilute human recombinant interleukin 6 into complete medium to give
2X stock solutions.
5. Add 50μl of the 2X dilutions of IL-6 to each well.
6. 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 100μl ONE-Glo detection reagent to each well and measure luminescence
following the recommended protocol. (Refer to the ONE-Glo Luciferase Assay
System Technical Manual, #TM292 for details).
