Inducible Tet-Off Regulatable System: An In Vitro Method to Modulate Gene Expression in Cultured Cells Using Tetracycline-Off System

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Transfection of the response plasmid expressing the gene of interest (GOI) into primary alveolar epithelial cells

1. Isolate type II alveolar epithelial cells from rat lungs.
2. Seed the cells at a density of 1 x 10⁶ cells/cm² in 100 mm Petri dishes with complete minimum essential medium (complete MEM). Complete MEM is MEM supplemented with 10% FBS, 0.08 mg/L tobramycin, Septra (3 µg/mL trimethoprim and 17 µg/mL sulfamethoxazole), 0.2% NaHCO₃, 0.01 M HEPES (pH = 7.3), and 2 mM L-glutamine. Culture the cells for 24 h at 37 °C in 5% CO₂ in a humidified incubator.
3. On the next day, place 500 µL of complete MEM without antibiotic in each well of a new 12 well plate and prewarm the plate at 37 °C for 30 min. During this step, it is important to use fetal bovine serum free of contaminating tetracyclines or with a level too low to interfere with inducibility.
4. Prepare 1.5 mL tubes containing the plasmid with the inducible GOI (GOI plasmid) and the regulatory vector (e.g., pTet-Off) by adding 1 µg of GOI plasmid and 1 µg of regulatory vector per well at RT. For coexpression experiments with RNA-binding proteins (RBP), 1 µg of a constitutive vector (e.g., pcDNA3) expressing the RPB of interest is added to the DNA mix (Figure 4).
5. Aspirate the medium and gently rinse the cells with PBS (without calcium and magnesium) prewarmed at 37 °C.
6. Add 5 mL of 0.05% trypsin prewarmed at 37 °C and incubate the cells until the cells are detached (2-4 min). Neutralize the trypsin by adding 10 mL of complete MEM without antibiotic.
7. Collect the cell suspension in a 50 mL tube, wash the Petri dish with 4 mL of medium to collect as many remaining cells as possible, and then centrifuge the cell suspension at 300 x g for 5 min.
8. Gently aspirate and discard the supernatant and resuspend the pellet in 1 mL of PBS. Count and calculate the number of cells using a hemocytometer.
9. Centrifuge the cells at 300 x g for 5 min. Gently aspirate the supernatant and resuspend the pellet in resuspension buffer at a concentration of 4 x 10⁷ cells/mL. Add the cells from the 1.5 mL tube prepared in step 2.4 at a concentration of 400,000 cells per well and gently mix them by pipetting up and down.
10. Place the tube in the electroporation device and fill it with 3.5 mL of electrolytic buffer.
11. Insert a gold-plated electrode tip into a pipette by completely pressing the piston. Gently mix the contents of the 1.5 mL tube and carefully aspirate the cells with the pipette. Be careful to prevent air bubbles from entering the tip, as this will cause electric arcing during electroporation and lead to decreased transfection efficiency.
12. Insert the pipette in the electroporation station until there is a clicking sound.
13. Select the appropriate electroporation protocol for alveolar epithelial cells, corresponding to a pulse voltage of 1,450 V and 2 pulses with a width of 20 ms, and press Start on the touchscreen.
14. Immediately after transfection, remove the pipette and transfer the cells to a well previously filled with complete MEM without antibiotic that has been prewarmed to 37 °C.
15. Repeat steps 1.11-1.14 for the remaining samples.
16. Gently shake the plate to spread the cells evenly over the well surface. Incubate the cells at 37 °C in 5% CO₂ in a humidified incubator. After 2 days, replace the medium with complete MEM with antibiotics.
17. Confirm the success of the transfection by observing the expression of eGFP under a fluorescence microscope or by flow cytometry using a control vector (Figure 1).
    NOTE: This step is optional and requires an additional transfection step using a different plasmid expressing eGFP, such as pcDNA3-EGFP.

2. Induction of the transcription inhibition of the GOI

NOTE: The cells can be pre-treated with the desired treatments before doxycycline induction to assess their impact on mRNA stability (Figure 3).

1. Prepare a doxycycline stock solution of 1 mg/mL in deionized water. Store the stock solution at -20 °C protected from light. Doxycycline, a tetracycline derivative, is used instead of tetracycline because it has a longer half-life (2x) than tetracycline. Moreover, a lower concentration of doxycycline is required for the complete inactivation of the tet operon.
   NOTE: Doxycycline could affect the mRNA expression of the endogenous GOI. To verify this, the effect of a 24 h treatment with doxycycline on alveolar cells should be tested to confirm the absence of any changes in GOI expression (Figure 2).
2. Prepare a fresh 1 µg/mL doxycycline solution in complete MEM 72 h posttransfection and warm it to 37 °C.
3. Replace the medium with 1 mL of complete MEM containing 1 µg/mL doxycycline per well to inhibit the transcription of the GOI.
4. Incubate multiple wells at 37 °C in 5% CO₂ for different amounts of time from 15 min-6 h to assess the mRNA half-life of the GOI.
5. At the end of the treatment, wash the cells with ice-cold PBS and lyse them with a commercially available phenol-chloroform RNA extraction kit by adding 500 µL of buffer per well and shaking the plate to homogenize the cells.
6. Isolate the RNA according to the manufacturer's protocol. Determine the RNA yield and purity by spectrophotometry at 230, 260, and 280 nm. RNA samples with 260:230 and 260:280 ratios of 1.8 and 2.0, respectively, are considered pure.
   NOTE: The protocol can be paused here.