The mifepristone-induced gene regulatory system (M I G R S ) is a system for regulating rake gene expression through the antiprogesterone drug, mifepristone (M f p ). The system utilizes a trans-activating chimeric protein that recombines a portion of the ligand-binding region of the human progesterone receptor (hPR-LBD), the D N A-binding region derived from the yeast Gal4 transcription factor, and the trans-activating region. In the absence of endogenous progesterone, M f p acts as a trans-activator that binds to activation sequences in the upstream region within the promoter of a sacrificial gene and initiates target gene transcription. Author: T. Friedman et al, Translated by W. Qin et al. This experiment is from "Gene Transfer".
Operation method
Use of MIGRS in cultured cells Move Use of MIGRS in cultured cells MATERIALS Reagents Dimethyl sulfoxide (DMSO, Sigma-Aldrich D8418) Ethanol (80%) Hank's Balanced Salt Solution (HBSS) Mifepristone (Mfp, RU-486; Sigma-Aldrich M8046) M fp powder is stored in a brown vial at 4°C. To avoid solidification of M fp by exposure to air, MfP may be equilibrated to room temperature by placing it in a closed container prior to weighing. Mfp-containing solutions should be protected from light as much as possible. Dissolve MfP powder in DMS0 to make a 10-2 mol/L stock solution. The stock solution can be stored at 1/2°C for up to 6 months. For cell culture experiments, dilute the stock solution with 80% ethanol to l0-2 mol/L as working solution. The working solution can be stored at 1-20℃ for 6 months. Add the working solution to the cell culture medium to achieve Mfp concentration of l0-8mol/L or l0-7mol/L. Add glutamate, antibiotics, 10 % fetal calf serum (FCS) and MEM. S F C S Cells Appropriate transfection reagents Instruments 37℃ CO2 cell incubator Methods 1. Transfect the cells with the vector. Transfection of plasmids a. Perform transfection primarily according to the appropriate manual for the transfection reagent. On the premise of ensuring that M IGRS and target genes are transfected into human cells by different plasmids, the ratio of these plasmids can be adjusted to control the level of basal versus induced expression of the target gene. For the GLVP switch protein, the molar ratio of the GLVP plasmid to the target gene plasmid is 1 : 10 to 1 : 2.5. For the GLp65 switch protein, the ratio of the two plasmids is 1 : 1, which results in a significantly lower basal expression. The strong constitutive promoter induced higher basal expression and a lower switch protein-to-target gene plasmid ratio. In conclusion, the choice of the two plasmid ratios was mainly empirical, with the aim of obtaining ideal basal and target gene induced expression. b. Immediately after transfection, cells were washed with HBSS and incubated for at least 3 h in M fp-free medium. c. Change the medium with pre-warmed cell culture medium containing 10-8 mol/L Mfp, 5%~10% SFCS. Transfection of recombinant viral vectors a. Determine the infection conditions according to the characteristics of the virus itself and the type of infected cells. HSV vectors, first generation adenoviral vectors, and HI> Ad vectors have been used in Vero, HeLa, and HePG2 cells, respectively. b. Immediately after transfection, wash the cells with HBSS to remove excess vector. c. Transfer the cells to normal medium and allow them to recover for 3 to 12 h. d. Finally, change the medium with SFCS cell culture medium containing l0-8 smol/LMfp, 5%~10% SFCS. 2. Cultivate cells in Mfp-containing medium for 24-72 h, the duration of which varies from cell to cell. 3 . Collect the cells and analyze the expression of target genes (Yeet al. 2002). Reagents mice Mifepristone (M f p , R U -486; Sigma-Aldrich M 8046) For intraperitoneal injection and gavage, dilute 10—2mol/L stock solution to 2X l0-4mol/L with sesame oil. Place this diluted solution at 4°C overnight to allow for stratification. Prior to injection, place the injection at room temperature in a place protected from light for approximately 1 h. The injection can be stored at 4°C for up to 3 weeks without affecting MFP activity. MFP can also be used for subcutaneous inoculation by encapsulating it in biodegradable slow-release pellets, which allows for stable and sustained injection of MFP. Technical support from Innovative Research of America (Sarasota, Florida) provided the best results. To calculate the amount of MFP encapsulated in a pellet, multiply the required daily dose of MFP (ug/kg) by the average body weight of the animal (kg) and multiply by the number of days of MFP administration. Plasmid Plasmid purification kit (large package) Polyvinylpyrrolidone or saline Sesame oil (Sigma-Aldrich S 3547) Instrumentation Electroporator with two parallel plate caliper electrodes (optional, see step 3) Methods 1. Purify the plasmid using the Plasmid Purification Kit (250 to 500ug), see kit instructions. 2. Resuspend the purified plasmid with 1mg/ml polyvinylpyrrolidone or saline. 3, inject 25~50ug of DNA into anesthetized mice tibialis anterior muscle or gastrocnemius muscle on both sides. Two parallel plate clamp electrodes can be used for electroporation and square wave pulse electrotransfection after injection to improve the efficiency of muscle transfection. The voltage of electroporation is usually 375 V/cm for two transfections at two 25-us square-wave pulses. 4. Since a slight inflammatory reaction may occur after plasmid injection, animals were allowed to recover for 4 d after injection or electroporation. 5. Calculate the amount of MFP according to the body weight of the animals, 250~300ug/kg, and dilute the MFP to 2 X l0-4mol/L with sesame oil. Administer the drug by intraperitoneal injection or by gavage. Alternatively, implantation of MFP-containing degradable slow-release particles can achieve the effect of continuous induction of target gene expression. 6. After administration of a specific dose of M f p , the time to analyze the expression of the target gene is chosen based on the half-life of the plasmid or target gene expression product in the cytoplasm. If the half-life is not known, the apparent kinetics of target gene expression should first be determined after M f p administration. Reagents For intraperitoneal injection and gastric gavage, dilute a 10-2mol/L reservoir solution to 2 X 0-4mol/L with sesame oil according to Protocol 1. The diluted solution is left overnight to allow for stratification. Prior to injection, allow the solution to rewarm at room temperature in a place protected from light for approximately 1 hour. The injection can be stored at 4°C for 3 weeks without affecting the MFP activity. Rodents Sesame Oil (Sgma-Aldrich S 3547) Instrumentation Positioning device or needle or syringe for tail vein injection (see Steps) Methods 1. Viral vectors encoding the MIGRS and target gene cassettes are injected into the animal by means of a localization device or systematic tail vein injection (Oligino et al., 1998; Burcin et al., 1999; Schillinger et al., 2005). The administration of large amounts of virus by both modes of delivery is capable of causing high basal expression. The level of basal and induced expression depends on a number of factors, including the ratio of virus particles to I U in the viral solution, the characteristics of the tissue being transfected, and the promoter used to initiate the expression of the switch protein. Suitable transfection conditions need to be worked out and it is recommended that all viral fluids be used as the initial dose. 2. Since the injection of viral vectors is often accompanied by local inflammatory reactions, allow animals to recover for 1~2 weeks after injection. 3. Dilute M f p with sesame oil to 2X 10-4mol/L and administer by intraperitoneal injection or gavage. 4. Select the appropriate time point to analyze the expression of the target gene. This should be determined by the half-life of the target gene in plasma or cytoplasm, or if the half-life is not known, the conditions need to be mapped out Reagents Mifepristone (M f p , R U -486; Sigma-Aldrich M 8046) For intraperitoneal injection and gavage, dilute the KT2mol/L reservoir solution to 2 X I0-4mol/L with sesame oil according to Protocol 1. Place this diluted solution at 4°C overnight to allow for stratification. Prior to injection, allow the solution to rewarm by placing it at room temperature in a place protected from light for approximately 1 hour. Mouse Sesame oil (Sigma-Aldrich S3547) Instrumentation Equipment for N o r t h e r n imprinting or quantitative reverse transcription of P C R (see step 4). METHODS 1. Creation of the first transgenic basal mouse line. The transfer of a carrier expressing a turn-on protein, which is initiated by a tissue-specific promoter, ensures that the target gene is ultimately expressed only in a specific tissue. 2 . Create a second transgenic basal mouse line. The target gene is transferred into a vector in which the target gene is regulated by a minimal promoter such as H SV thymidine kinase or adenotoxic EIBTATA and is located after the multicopy Gal4 UAS. 3. Activate the expression of the target gene (the same as in Scheme 3, where MIGRS is used in the gene therapy model). Dissolve 330ug/kg M f p in sesame oil (2 X 10-4mol/L ) and administer by intraperitoneal injection or gavage. Subcutaneous implantation of degradable slow-release pellets containing Mfp can be used to obtain stable, long-term expression of the target gene. 4. Analyze the expression characteristics of the switch protein in each transgenic basal mouse line by Northern blotting or reverse transcription PCR. In general, high levels of switch protein expression can lead to high levels of maximal induced expression of the target gene, but at the same time, it also results in high basal expression. 5 . When it is clear that the corresponding genes are properly expressed in the two basal mouse lines, the two are crossed to obtain double transgenic mice that can express both switch proteins and MFP-induced, tissue-specific target genes. Similar to the transfection of gene therapy models, the basal and induced expression levels of target genes in transgenic models depend on the expression of switch proteins in specific tissues. In turn, the level of switch protein expression is affected by the corresponding promoter, chromosomal location, and the number of switch protein expression cassettes in the target specific transgenic mouse line. For more product details, please visit Aladdin Scientific website.
The appropriate culture medium for the cultured cells.
Mifepristone (M f p , R U -486; Sigma-Aldrich M 8046)
Allow to rewarm. The injection can be stored at 4°C for up to 3 weeks without affecting M fp activity. Continuous and stable transfection was achieved by subcutaneous implantation of slow-release degradable particles containing Mfp. The best results were obtained with the technical support of Innovative Research of America (Sarasota, Florida). To calculate the amount of Mfp encapsulated in a pellet, multiply the required daily dose of Mfp (ug/kg) by the average body weight of the animal (kg) and multiply by the number of days of Mfp administration.