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

Overview

This video describes the method of regulating gene expression in cultured cells by transfection with a plasmid containing a gene of interest and a regulatory plasmid. The expression of this plasmid can be controlled by tetracycline or its synthetic analogs, making this system a useful model to study regulatable gene expression.

Protocol

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.

Representative Results

Figure 1: Efficiency of the transfection of alveolar epithelial cells in primary culture by pipette electroporation. Primary alveolar epithelial cells were transiently transfected with 2 µg of a pcDNA3 plasmid (empty, clones #1 and #2) that expressed or did not express GFP protein. Transfection efficiency was assessed 48 h following transfection by (A) fluorescence microscopy or (B) flow cytometry. One-way ANOVA and Bonferroni post hoc test; *p < 0.001 vs. empty. Cells from at least four different rats (n ≥ 4) were used for each experimental condition. Scale bar = 200 µm. 

Figure 2: Modulation of endogenous αENaC mRNA by doxycycline in alveolar epithelial cells. Alveolar epithelial cells were treated with 1.0 µg/mL doxycycline for a period of 1-24 h. The expression of αENaC mRNA was quantified by quantitative RT-PCR and presented as the expression of αENaC mRNA ± SEM compared to that in untreated cells (Ctrl; t = 0) after normalization according to β-actin expression (one-way ANOVA, n = 4). Doxycycline did not modulate endogenous αENaC mRNA over time. Previously published as Figure S4 in Migneault et al.

Figure 3: Modulation of V5-αENaC mRNA stability by different cellular and inflammatory stresses. Primary alveolar epithelial cells were transiently cotransfected with the pTet-Off plasmid and the pTRE-tight plasmid encoding αENaC cDNA bearing a V5 epitope upstream of its open reading frame and complete 3' UTR sequences. The cells were pretreated for 30 min with 1.0 µM cycloheximide (CHX) (A) or 15 µg/mL LPS (B) or for 5 h with 100 ng/mL TNF-α (C), followed by treatment with 1.0 µg/mL doxycycline for a period of 15-120 min. Expression of V5-αENaC mRNA was measured by quantitative RT-PCR and presented as the percentage ± SEM of V5-αENaC mRNA expression in untreated cells (t = 0) after normalization according to the expression of tTA-Ad. Cells from at least three different rats (n ≥ 3) were used for each experimental condition. (D) The half-life (t_1/2) of V5-αENaC mRNA in treated cells was compared to the half-life of mRNA in cells (Ctrl). The half-lives were measured according to the rate constant (K) of the V5-αENaC mRNA degradation curve using the equation t_1/2 = ln 2/K and then expressed as min ± SEM (one-way ANOVA test and Bonferroni post hoc test; *p < 0.01 vs. control; n ≥ 3). Adapted from Figure 36 previously published in Migneault, F.

Figure 4: Posttranscriptional modulation of V5-αENaC mRNA by the RNA-binding proteins hnRNPK, Dhx36, and Tial1. (A) Primary alveolar epithelial cells were cotransfected with the pTRE-tight plasmid encoding V5-αENaC mRNA along with an expression vector for the Dhx36, hnRNPK, or Tial1 RBPs and the pTet-Off plasmid. V5-αENaC mRNA expression was quantified by RT-qPCR 72 h posttransfection and expressed as the percentage ± SEM of V5-αENaC mRNA expression compared to that in cells transfected with an empty vector (pcDNA3) after normalization according to the expression tTA-Ad. Overexpression of Dhx36 and Tial1 significantly inhibited V5-αENaC mRNA expression, whereas overexpression of hnRNPK had no effect. *p < 0.05 according to the Kruskal-Wallis test and Dunn's post hoc test compared to empty vector; n ≥ 3 samples from different animals were tested in duplicate for each experimental condition. (B) The proximal portion of the αENaC 3' UTR was deleted by cloning the distal region of the 3' UTR next to the αENaC stop codon in the pTRE-tight plasmid (V5-αENaC-Del5). (C) Primary alveolar epithelial cells were cotransfected with V5-αENaC or V5-αENaC-Del5 in the pTRE-tight vector along with the pTet-Off plasmid and the expression vector for Dhx36 or Tial1 RBP overexpression. V5-αENaC mRNA expression was quantified by RT-qPCR 72 h posttransfection and expressed as the percentage ± SEM of V5-αENaC mRNA expression compared with that in cells transfected with an empty vector (pcDNA3) after normalization according to the expression of tTA-Ad. Overexpression of Dhx36 and Tial1 had no effect on V5-αENaC-Del5 mRNA expression. *p < 0.05 according to the Kruskal-Wallis test and Dunn's post hoc tests upon comparison of the experimental vectors to the empty vector; #p < 0.05 according to the Mann-Whitney U-test upon comparison of the experimental vectors to the complete 3' UTR mutant; n ≥ 6 for each experimental condition. Adapted from Figures 5 and 7 previously published in Migneault et al.

Materials

Name	Company	Catalog Number	Comments
Bright-LineHemacytometer	Sigma-Aldrich	Z359629
DM IL LED Inverted Microscope with Phase Contrast	Leica
Dulbecco’s Phosphate-buffered Saline (D-PBS), without calcium and magnesium	Wisent Bioproducts	311-425-CL
Doxycycline hyclate	Sigma-Aldrich	D9891-1G
MEM, powder	Gibco	61100103
MSC-Advantage Class II Biological Safety Cabinets	ThermoFisher Scientific	51025413
Neon Transfection System 10 µL Kit	Invitrogen	MPK1025
Neon Transfection System Starter Pack	Invitrogen	MPK5000S
pcDNA3 vector	ThermoFisher Scientific	V790-20
pcDNA3-EGFP plasmid	Addgene	13031
pTet-Off Advanced vector	Takara Bio	631070
pTRE-Tight vector	Takara Bio	631059
Purified alveolar epithelial cells	n.a.	n.a
QIAEX II Gel Extraction Kit	QIAGEN	20021
QIAGEN Plasmid Maxi Kit	QIAGEN	12162
QIAprep Spin Miniprep Kit	QIAGEN	27104
QuantStudio 6 and 7 Flex RealTime PCR System Software	Applied Biosystems	n.a.
Tet System Approved FBS	Takara Bio	631367