pGL4.48載體簡介
The pGL4.48[luc2P/SBE/Hygro] Vector contains three copies of a Smad-binding element (SBE) 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 pGL4.48[luc2P/SBE/Hygro] vector is used to measure activation
of the SBE in HEK293 cells upon treatment with TGF-β1. 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)
0.05% Trypsin-EDTA (Life Technologies Cat.# 25300)
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])
Opti-MEM I (Life Technologies Cat.# 31985)
FuGENE HD Transfection Reagent (Cat.# E2311)
Human TGF-β1 (Sigma Cat.# T7039-2UG)
BSA (Sigma Cat.# B4287)
ONE-Glo Luciferase Assay System (Cat.# E6120)
HEK293 cells
實驗流程
Day 1: Reverse Transfection
Preparation of Cells
1. Grow HEK293 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 233 x g for 5 minutes in a swinging-bucket rotor.
Resuspend in complete medium at a concentration of 2 × 105 cells/ml.
Preparation of Lipid:DNA Mixture
1. Dilute pGL4.48[luc2P/SBE/Hygro] DNA to 10ng 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 and Luminescence Measurement
1. Resuspend human TGF-β1 (hTGF-β1) to 50μg/ml in water. Dilute 50X into DPBS +
2 mg/ml BSA to give a 1μg/ml solution, and then serially dilute into the same buffer
to give 10X stocks.
2. Add 10μl of the 10X dilutions of hTGF-β1 to each well and incubate for 3 hours in a
in a 37°C, 5% CO2 incubator.
3. Remove plates from the 37°C, 5% CO2 incubator and allow them to cool to room
temperature for approximately 15 minutes.
4. Add 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).
