pGL4.42載體介紹
The pGL4.42[luc2P/HRE/Hygro] Vector contains four copies of a hypoxia response element (HRE) 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 pGL4.42[luc2P/HRE/Hygro] Vector is used to measure
activation of the HRE in HEK293 cells upon treatment with 1,10-phenanthroline. 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.
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)
Opti-MEM I (Life Technologies Cat.# 31985)
FuGENE HD Transfection Reagent (Cat.# E2311)
DMSO (Sigma Cat.# D2650)
1,10-phenanthroline (Sigma Cat.# 131377))
Dual-Glo Luciferase Assay System (Cat.# E2940)
HEK293 cells
pGL4.75[hRluc/CMV] Vector (Cat.# E6931)
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 1 × 105 cells/ml.
Preparation of Lipid:DNA Mixture
1. Dilute pGL4.42[luc2P/HRE/Hygro] and pGL4.75 [hRluc/CMV] Renilla luciferase
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: Cell Treatment and Luminescence Measurement
1. Dissolve 1,10-phenanthroline to a final concentration of 50mM in DMSO. Serially
dilute this solution using DMSO to give a range of concentrated stock solutions
(500X). Dilute each concentrated stock solution using Opti-MEM I to give a range
of dilute stock solutions (10X). Add 10μl of the 10X stocks to each well.
2. Incubate for 5 hours in a 37°C, 5% CO2 incubator.
3. Remove plates from the 37°C, 5% CO2 incubator. Allow plates 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).
