The overall goal of this experiment is to modulate transgene expression by the administration of the chemical inducer tebufenozide. This method can help answer key questions in the cell and molecular biology field, such as when you need to stimulate the expression of a gene of interest at a desired time with a desired strength. The main advantage of this technique is that by combining all the required elements for transgene expression within a single vector, transgene expression can be stimulated by the administration of the chemical inducer tebufenozide.
After subcloning the EGFP or ANKRD13A transgenes according to the text protocol, prepare four cell culture dishes containing freshly passaged HEK 293 cells in five milliliters of DMEM supplemented with five to 10%FBS and antibiotics, which will hereafter be referred to as culture medium. Maintain the cells at 37 degrees Celsius. When the cells reach 50 to 60%density, add three microliters of transfection reagent to 200 microliters of diluent containing the DNA.
After incubating the reaction at room temperature for 30 minutes, add the mixture to the HEK 293 cells. Use two dishes for transfection, and leave the other two untreated as controls. Incubate the cells at 37 degrees Celsius for 12 to 24 hours.
Following the incubation, add Teb to one of the transfected samples and to one of the non-transfected controls. Leave the other two samples untreated as experimental controls. Incubate the plates for 12 to 48 hours.
If using the EGFP insert, analyze EGFP expression under a fluorescent microscope. Otherwise, to harvest the cells for immunoblotting, use ice-cold PBS to rinse the cells twice. Then, use a cell scraper to remove them from the plate, and transfer them into a 15-milliliter conical tube.
Next, spin the tubes at 21, 000 times g and 18 to 25 degrees Celsius for two minutes. Then, after discarding the PBS, add 0.5 milliliters of ice-cold mammalian protein extraction reagent, or M-PER, and protease inhibitor to the pellet. Resuspend the cells with several rounds of pipetting.
Then, incubate the cell suspension at 18 to 25 degrees Celsius and room temperature for 15 minutes. Spin the cell lysate at 21, 000 times g and four degrees Celsius for 10 minutes. Then, transfer the aqueous supernatant into a fresh 1.5-milliliter microcentrifuge tube, and analyze it by immunoblotting with the appropriate antibodies.
To quench the Teb stimuli, remove the Teb containing culture medium from the cells, and use pre-warmed PBS to rinse the cells twice. Then, add one milliliter of 0.25%trypsin/EDTA, and incubate the cells at 37 degrees Celsius for one minute. Add 10 milliliters of pre-warmed, Teb-free culture medium to the cells, and use a cell scraper to harvest them.
Transfer the cells to a 15-milliliter conical tube. Then, spin the cells at 21, 000 times g and 18 to 25 degrees Celsius for two minutes. Resuspend the cells in 50 milliliters of pre-warmed, Teb-free culture medium, and transfer five milliliters of the cells into three 60-millimeter fresh cell culture dishes.
Incubate the cells at 37 degrees Celsius for 24, 48, and 72 hours, respectively. Finally, harvest the cells at different time points, and measure the expression levels of the transgene using immunoblotting. In this experiment that evaluated the activity and leakiness of pEUI-plus-EGFP, mock vector transfectants showed no fluorescent signal regardless of Teb treatment.
However, pEUI-plus-EGFP transfectants became progressively sensitized to the increasing amount of Teb. More importantly, pEUI-plus-EGFP transfectants not treated with Teb did not elicit any detectable EGFP signals. FLAG-tagged ANKRD13A was used to further validate the responsiveness of pEUI-plus to Teb.
As shown in this western blot using anti-FLAG antibodies, transfected HEK 293 cells exhibited strong ANKRD13A expression following a 24-hour treatment with Teb. In addition, depletion of Teb demonstrated the reversibility of the pEUI-plus gene switch, as ANKRD13A was no longer detectable in cells cultured without Teb. Once mastered, this technique can be done in one hour if it is performed properly.
While attempting this procedure, it's important to remember to carry out almost all the experiment under germ-free conditions. Following this procedure, other methods, like the Tet-On or Tet-Off system can be applied to differentially express two distinct transgenes. After its development, this technique paved the way for researchers in the field of cell biology to explore molecular physiology at the cellular level.
After watching this video, you should have a good understanding of how to use the pEUI-plus gene switch vector for the purpose of inducing transgene expression at a desired time with a desired strength. Don't forget that working with tebufenozide can be hazardous and precautions, such as wearing a lab coat, should always be taken while performing this procedure.
