pGL4.44载体简介
The pGL4.44[luc2P/AP1 RE/Hygro] Vector contains six copies of an AP1 response element (AP1 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 pGL4.44[luc2P/AP1 RE/Hygro] Vector is used to measure
activation of the AP1 RE in HEK293 cells upon treatment with PMA. The pGL4.75 Vector
(encoding Renilla luciferase) is used as a normalization control. 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
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])
Opti-MEM I (Life Technologies Cat.# 31985)
FuGENE HD Transfection Reagent (Cat.# E2311)
PMA (Cat.# V1171)
Dual-Glo Luciferase Assay System (Cat.# E2940)
HEK293 cells
pGL4.75[hRluc/CMV] Vector (Cat.# E6931)
实验流程
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 1 × 105 cells/ml.
Preparation of Lipid:DNA Mixture
1. Dilute pGL4.44[luc2P/AP1 RE/Hygro] and pGL4.75 [hRluc/CMV] Renilla luciferase
control vector constructs in a 10:1 mass ratio, respectively, 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 × 105 cells/ml cell suspension. Mix by
pipetting.
4. Plate 100μl per well into a solid, white 96-well plate (Corning Cat.# 3917).
5. Incubate for 24 hours in a 37°C, 5% CO2 incubator.
Day 2: Medium Replacement
1. Aspirate medium and replace with 75μl DMEM + 0.1% FBS.
2. Incubate for 17 hours in a 37°C, 5% CO2 incubator.
Day 3: Cell Treatment and Luminescence Measurement
1. Dissolve PMA in DMSO to a final concentration of 10mM. Serially dilute this
solution in DMSO to give a range of concentrated stock solutions (1,000X). Dilute
each concentrated stock solution using Opti-MEM I to give a range of dilute stock
solutions (16X). Add 5μl of dilute stock solution to the existing 75μl of medium per
well, covering a final concentration range of PMA from 1pM to 1μM.
2. Incubate for 6 hours in a 37°C, 5% CO2 incubator.
3. Remove plates from the incubator and allow them to cool to room temperature for
approximately 15 minutes.
4. Add Dual-Glo Luciferase Assay System detection reagents, and measure luminescence
following the recommended protocol (Refer to the Dual-Glo Luciferase
Assay System Technical Manual, #TM058 for details).
