Inducible Tet-Off Regulatable System: An In Vitro Method to Modulate Gene Expression in Cultured Cells Using Tetracycline-Off System
Transkript
Seed the cells at a 1 x 106 cells/cm2 in 100-millimeter Petri dishes, in the presence of complete minimum essential medium. Culture for 24 hours at 37 degrees Celsius in 5% carbon dioxide with humidity. The next day, add 500 microliters of complete medium without antibiotic to each well of a new 12-well plate, and prewarm the plate at 37 degrees Celsius for 30 minutes.
During this incubation, add 1 microgram of the response plasmid and 1 microgram of regulatory vector to one 1.5-milliliter tube per well. Wash the alveolar epithelial cells with 10 milliliters per well of 37 degrees Celsius PBS without calcium or magnesium, and treat the cells with 5 milliliters per well of 37 degrees Celsius 0.05% trypsin for two to four minutes in the cell culture incubator.
When the cells have detached, neutralize the trypsin with 10 milliliters per well of complete medium without antibiotic, and collect the cells into a new 50-milliliter tube. Wash the dish with 4 milliliters of fresh medium to collect any remaining cells, and sediment the cells by centrifugation.
Resuspend the pellet in 1 milliliter of PBS for counting, and centrifuge the cells again. Re-suspend the pellet at a density of 4 x 107 cells/mL of resuspension buffer, and add 4 x 105 cells to each tube of plasmid and vector with gentle mixing. Place 1 tube in the electroporation device and fill the tube with 3.5 milliliters of electrolytic buffer.
Fully depress the piston to insert a gold-plated electrode tip into a pipette and gently mix the tube contents. Carefully aspirate the cells with a pipette, and insert the pipette into the electroporation station until a clicking sound is heard. Select the appropriate electroporation protocol for alveolar epithelial cells, and press Start on the touch screen.
Immediately after the transfection, remove the pipette and transfer the cells into one well of the prewarmed 12-well plate. When all of the cells have been electroporated and plated, place the plate in the cell culture incubator, replacing the supernatant in each well with complete medium with antibiotics after two days. The success of the transfection can be confirmed by the expression of EGFP, as observed by fluorescence microscopy or flow cytometry using a control vector.
To inhibit the transcription of the gene of interest, 72 hours post-transfection, replace the supernatant with 1 milliliter per well of complete medium supplemented with 1 microgram per milliliter of freshly prepared doxycycline. To assess the mRNA half-life of the gene of interest, return the plate to the cell culture incubator from 15 minutes to 6 hours.
Washing one well with 1 milliliter of ice-cold PBS, before lysing the cells with 500 microliters of lysis buffer from a commercially available phenol-chloroform RNA extraction kit, and gentle shaking at each experimental time point. Then, isolate the RNA according to kit instructions, and determine the RNA yield and purity by spectrophotometry at 230, 260, and 280 nanometers.
