pGL4.52載體簡介
The pGL4.52[luc2P/STAT5 RE/Hygro] Vector contains five copies of a STAT5 response element (STAT5 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 pGL4.52[luc2P/STAT5 RE/Hygro] Vector is used to measure
activation of the STAT5 RE in Ba/F3 cells upon treatment with mIL-3. 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
Dulbecco’s PBS (DPBS; Life Technologies Cat.# 14190)
Complete medium (RPMI Medium [Life Technologies Cat.# 22400]
+ 10% heat-inactivated FBS [Life Technologies Cat.# 10082-139]
+ 1 ng/ml mIL-3 [R&D Systems Cat.# 403-ML])
Opti-MEM I (Life Technologies Cat.# 11058)
FuGENE HD Transfection Reagent (Cat.# E2311)
mIL-3 (R&D Systems Cat.# 403-ML)
BSA (Proliant Cat.# 68700)
ONE-Glo Luciferase Assay System (Cat.# E6120)
Ba/F3 cells
實驗流程
Day 1: Reverse Transfection
Preparation of Cells
1. Grow Ba/F3 cells in suspension in complete medium [RPMI Medium + 10%
heat-inactivated FBS + 1ng/ml mIL-3].
2. Quantify cells and dilute them in complete medium to 1 × 105 cells/ml.
Preparation of Lipid:DNA Mixture
1. Dilute pGL4.52[luc2P/STAT5 RE/Hygro] Vector to 10ng total DNA/μl in Opti-MEM I.
2. Add FuGENE HD to a 6:1 lipid:DNA ratio and mix gently. Incubate at room
temperature for 15 minutes.
3. Dilute lipid:DNA mixture 20-fold with 1 × 105 cells/ml cell suspension and mix by
inversion.
4. Place cells in a flask and incubate for 18–24 hours in a 37°C, 5% CO2 incubator.
Day 2: Plating, Cell Treatment and Luminescence Measurement
Plating Cells
1. Pellet the cells by centrifugation at 200 × g for 5 minutes in a swinging-bucket rotor.
Wash once with DPBS and spin again.
2. Resuspend cells in Opti-MEM I to 1 × 106 cells/ml.
3. Plate 100μl per well to a solid, white 96-well plate (Corning Cat.# 3917).
4. Incubate for 4 hours in a 37°C, 5% CO2 incubator.
Cell Treatment
1. Resuspend mIL-3 to 0.1 mg/mL in DPBS + 0.1% BSA. Make serial dilutions into
Opti-MEM I to make 10X stocks.
2. Add 10μl of the 10X stocks of mIL-3 to each well and incubate for 4 hours in a 37°C,
5% CO2 incubator.
Luminescence Measurement
3. Remove plates from the 37°C, 5% CO2 incubator and allow them to cool to room
temperature for approximately 15 minutes.
4. Add 100μl of ONE-Glo Luciferase Assay System detection reagent to each well and
measure luminescence following the recommended protocol
