The RheoSwitch system is an efficient gene regulatory system based on the ecdysone receptor, which is activated by synthetic small molecule ligands. The receptor consists of a two-hybrid heterodimerization of the ecdysone receptor protein and the R X R protein, which is transcriptionally activated from the corresponding promoter in the presence of the ligand. The RheoSwitch system functions in animal cells, yeast, and plants. Author: T. Friedman et al, Translated by Wei-Qing et al. This experiment is from "Gene Transfer".
Operation method
Synthesized small molecule ligands R S L l induced gene regulation in vitro Move Synthesized small molecule ligands R S L l induced gene regulation in vitro Material Reagents Calf serum (Invitrogen) Cells to be transfected Dimethyl sarcoside (DMSO; Sigma-Aldrich) Ligand: R S L l (5 mmol/L in DMSO, stable at room temperature, MW 382.5) ( New EnglandBioLabs) Liposome 2000 (Invitrogen) Sudden light detection system (Bright-Glo; Promega) Nucleic Acids Inducible expression vector: pNEBR-Xl (NewEnglandBioLabs) Standard Plasmid: pRL-CMV (optimal, see Step 2a) (Prom ega) Receptor vector: pNEBR-Rl (NewEnglandBioLabs) Bacteria (XLl-Bkie or others) containing the plasmid are cultured in L B medium with 100ug/ml ampicillin. To de-endotoxify the plasmid for transfection, column purification (e.g., Plasmid Extraction Kit from QIAGEN) or CsCl gradient methods are appropriate. Opti-MEMI medium (Invitrogen) or a serum-free medium is suitable. PLB lysate (Promega) TRIZOL Reagent (Invitrogen) or RNeasy Kit (QIAGEN) Instrumentation Cell culture plate (48-well plate in this case), 96-well opaque assay plate (VWR or Fisher Scientific) Microcentrifuge tubes (1.7 ml) Microplate chiller (Dynex Technologies, Inc.) for sudden light detection Planar oscillator Methods Transfection The RheoSwitch System is compatible with a variety of transfection reagents including Liposome 2000 (Invitrogen), F u G E N E 6 (Gene Therapy System), SuperFect (Q I A G E N ), and Calcium Phosphate Precipitates. Electroporation and nuclear transfection techniques (Amaxa Biosystems) can be used. Transfection can be performed on a large scale in a variety of cell culture plates (96-well, 48-well, 24-well). Here, Liposome 2000 is used to transfect cells in 48-well plates with p NEBR-Rl as the receptor vector and p NEBR-Xl containing insect fluoresceinase as the inducible expression vector. The program can be modified to suit the application. 1. If adherent cells are used, inoculate the cells the day before transfection in 250 M1 medium without antibiotics so that the cells are 80 % to 90 % confluent at the time of transfection. The use of Lipofectamine 2000 in antibiotic-containing medium can cause cytotoxicity. 2 . Prepare 6 wells of transfection mixture as described below, repeat each transfection experiment and statistically analyze the final results. a. Dilute the RheoSwitch System Plasmid in a 1.7 ml microcentrifuge tube with 160 ul of Opti-MEMI (or serum-free medium), 500 ng of Receptor Carrier (pNEBR-R l) and 1.5 Mg of Inducible Expression Carrier (pNEBR-X l). b. Dilute the plasmid with the RheoSwitch System Plasmid. c. Dilute the RheoSwitch System Plasmid with the RheoSwitch System Plasmid. If desired, a control reporter gene driven by a constitutive promoter, such as renal fluorokinase or lacZ, can be added and can be used to correct transfection efficiency and detection between wells. Add 50ng of Correction Plasmid to a microcentrifuge tube. b. Add 6^1 of Lipofectamine 2000 to 160ul of OptiMEMI (or serum-free medium) in another 1.7ml microcentrifuge tube. c. After 5 min at room temperature, combine the diluted DNA and diluted Lipofectamine 2000, gently drop and allow to stand for 20 min. 3 . Add 50jlJ of transfection mixture per well (6 total). 4 . The cell plate was taken to the incubator. To optimize transfection efficiency, the optimal amount of DNA and the ratio of DNA to Lipofectamine2000 needs to be explored for different cell lines. Ligand induced gene expression. Depending on the level of gene induction to be obtained, a ligand concentration test is performed as follows to determine the optimal amount of ligand. 5. 5 mmol/L R S L l (in D M S O ) storage solution is diluted into different working solutions. Suggested working solution concentrations are 0.8, 4, 20, 100, and 500umol/L . 6. Dilute each set of working solutions 1000-fold in cell plate well culture solution (to form concentrations of 0.8, 4, 20, 100, and 500 nmol/L RSU ) and mix well. Prepare the required volume of ligand culture solution. 7 . After 4~6 h of transfection, remove the medium from the transfected cell plate wells and add 250ul of culture medium containing different concentrations of ligands. 8 . Take the cell plate to the incubator. The current transfection mixture does not affect the ability of ligand-induced genes to be expressed, and, in most cases, it is not necessary to remove the culture medium containing the transfection mixture from the cell plate wells. This is particularly useful when a large number of transfection assays are performed. In this case, the above scheme may be modified as follows. 9 . Prepare a culture solution containing two times the desired concentration of ligand (e.g., 0.16 nmol/L, 8 nmol/L, 40 nmol/L, 200 nmol/L, lumol/L RSLl Ligand). 10. Repeat transfection as described in steps 1 and 2 above. 11. After inoculation of the transfection mixture to form the DNA-Lipofectamine 2000 complex, remove the culture medium from the cell plate wells and add 75ul of fresh culture medium. 12. Dispense 50ul of transfection mixture per cell plate well and gently shake the cell culture plate (125ul of culture medium per well). 13. Add 125 ml of culture medium containing 2-fold dilution concentration of ligand prepared in step 9. The ligand will be diluted to half the original concentration. It is not necessary to incubate the cells between steps 12 and 13. Detection of induced gene expression Induced gene expression can be detected at the mRNA level within a few hours after ligand treatment or at the protein level using a highly sensitive method such as Chemiluminescence or Green Fluorescent Protein (GFP). After 24 h of ligand treatment, the expression is sufficiently large, and at 48-72 h, the expression reaches its maximum. There is no need to change the culture medium or add more ligands (unless the culture medium is changed). 15. Detect inducible genes at the mRNA 7jC level, isolate total RNA from cells and perform Northern hybridization, or reverse transcribe RNA and perform real-time fluorescence quantitative PCR. The format of the plates used for transfection is selected according to the method of RNA isolation. To isolate RNA, use TRIZOL reagent or RNeasy Kit. For more product details, please visit Aladdin Scientific website.
Ampicillin (100 mg/ml aqueous solution)
H . Gently shake the cell culture plate and take it to the incubator.
