pGL4.49載體簡介
The pGL4.49[luc2P/TCF-LEF RE/Hygro] Vector(a–c) contains eight copies of a TCF-LEF response element (TCF-LEF 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 pGL4.49[luc2P/TCF-LEF RE/Hygro] vector is used to
measure activation of the TCF-LEF RE in HEK293 cells upon treatment with mWnt3a or
LiCl. 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% Tryspin-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])
Charcoal-stripped FBS (Life Technologies Cat.# 126776-011)
Opti-MEM I (Life Technologies Cat.# 31985)
FuGENE HD Transfection Reagent (Cat.# E2311)
mWnt3a (R&D Systems Cat.# 1324-WN)
BSA (Proliant cat# 68700)
LiCl (Sigma Cat.# 40113)
ONE-Glo Luciferase Assay System (Cat.# E6110)
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. Quantify cells and dilute in complete medium to 1.5 × 105 cells/ml.
Preparation of Lipid:DNA Mixture
1. Dilute pGL4.49[luc2P/TCF-LEF RE/Hygro] Vector 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 1.5 × 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: Medium Replacement and Cell Treatment
1. Resuspend mWnt3a to 100μg/ml in DPBS + 1 mg/ml BSA. Dilute mWnt3a to 3μg/ml
and then serially dilute into PBS/BSA to give a range of 10X stock solutions. Serially
dilute 1M LiCl into water to give a range of 10X stock solutions.
2. Remove existing medium from cells and replace with 90μl of DMEM + 0.5%
charcoal-stripped FBS per well.
3. Add 10μl of the 10X dilutions of mWnt3a or LiCl.
4. Incubate for 8 hours in a 37°C, 5% CO2 incubator.
Day 3: Luminescence Measurement
1. Remove plates from the incubator and allow them to cool to room temperature for
approximately 15 minutes.
4. Add ONE-Glo Luciferase Assay System detection reagent, and measure
luminescence following the recommended protocol (refer to the ONE-Glo
Luciferase Assay System Technical Manual, #TM292 for details).
