pGL4.45載體簡介
The pGL4.45[luc2P/ISRE/Hygro] Vector contains five copies of an interferon-stimulated response element (ISRE) 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 pGL4.45[luc2P/ISRE/Hygro] Vector is used to measure
activation of the ISRE in U2OS cells upon treatment with interferon-alpha. 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
[HyClone Cat.# SH30070.03])
0.05% Tryspin-EDTA (Life Technologies Cat.# 25300)
Dulbecco’s PBS (DPBS; Life Technologies Cat.# 14190)
Opti-MEM I (Life Technologies Cat.# 31985)
FuGENE HD Transfection Reagent (Cat.# E2311)
Interferon-alpha (IFN-alpha; EMD Cat.# 407294) )
ONE-Glo Luciferase Assay System (Cat.# E6120)
U2OS cells
實驗流程
Day 1: Reverse Transfection
Preparation of Cells
1. Grow U2OS cells in complete medium (McCoy’s 5A + 10% FBS). Wash with
DPBS and treat with one volume of 0.05% trypsin-EDTA. Resuspend the cells in
four volumes of complete medium.
2. Quantify cells and dilute in complete medium to 2 × 105 cells/ml.
Preparation of Lipid:DNA Mixture
1. Dilute pGL4.45[luc2P/ISRE/Hygro] DNA 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 2 × 105 cells/ml cell suspension. 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: Cell Treatment
1. Serially dilute IFN-alpha into complete medium to give 10X stock solutions.
2. Add 10μl of the 10X dilutions of IFN-alpha to each well.
3. Incubate for 16 hours in a 37°C, 5% CO2 incubator.
Day 3: Luminescence Measurement
1. Remove plates from the 37°C, 5% CO2 incubator. Allow plates to cool to room
temperature for approximately 15 minutes.
2. Add 100μl ONE-Glo Luciferase Assay System 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).
