Efficient Transcriptionally Controlled Plasmid Expression System for Investigation of the Stability of mRNA Transcripts in Primary Alveolar Epithelial Cells

All animal procedures were conducted according to the guidelines of the Canadian Council on Animal Care and were approved by the Institutional Animal Care Committee of the Research Center of Centre Hospitalier de l'Université de Montréal (CRCHUM).

1. Design and generation of the response plasmid expressing the gene of interest (GOI)

1. Use an inducible tetracycline-off vector, such as pTRE-Tight.
2. Analyze the sequence of the GOI and the multiple cloning site (MCS) of the vector to identify the restriction sites in the MCS that are not present internally in the GOI.
3. Isolate primary alveolar epithelial cells from male Sprague-Dawley rat lungs as described previously^19,20.
4. Purify the total RNA from the alveolar epithelial cells by RNA extraction using a standard method, such as phenol/chloroform extraction or the use of silica-based RNA spin columns.
5. Reverse-transcribe the mRNA into complementary DNA (cDNA) using oligo(dT) and high-fidelity reverse transcriptase.
6. Use high-fidelity Taq polymerase and standard overlap PCR techniques to flank the GOI with two selected restriction enzyme recognition sites using designed primers.
   1. Have the forward primer contain a Kozak consensus ribosome binding site²¹ to improve expression levels to study protein expression in parallel with mRNA stability. A sequence encoding the V5 epitope upstream of the GOI must be included to distinguish the expression of the transfected GOI from endogenous expression (Table 1).
   2. Have the reverse primer contain a polyadenylation signal after the stop codon.
7. Mutants can be generated by sequential deletion to study the effects of different 3' UTR regions on the stability of the mRNA of the GOI using reverse primers encoding a polyadenylation site that gradually deletes the 3' end of the GOI 3' UTR (Figure 6). Alternatively, PCR-directed mutagenesis can be used to target a specific region of interest in the 3' UTR²².
8. Digest the inducible vector and the insert with the previously chosen restriction enzymes at the appropriate incubation temperature for 1 h, followed by treatment with phosphatase during the vector reaction for 30 min to avoid self-ligation.
9. Separate the digested vector and insert segments by electrophoresis in a 1-1.5% agarose gel (concentration depending on the size of the insert).
10. Using a blade and a UV light, collect the DNA fragments containing the desired insert and vector to be ligated.
    NOTE: The protocol can be paused here.
11. Purify the collected segments from the agarose gel using a silica-based PCR purification kit and measure the concentration by spectrophotometry at 260 nm.
12. Ligate the GOI and the inducible vector with T4 DNA ligase using a vector:insert molar ratio of 1:3 to increase the probability of ligation. Incubate the reaction at room temperature (RT) for 3 h.
13. Transform the ligation reaction into competent E. coli (DH5α).
    1. Add 1-10 ng of vector and gene to a tube containing 100 µL of competent cells. Incubate the cells on ice for 30 min and then heat-shock cells at 42 °C for 45 s. Place the tube on ice for 2 min and add 900 µL of RT LB medium. Incubate the cells for 1 h at 37 °C with shaking at 225 rpm.
    2. Spread 100 µL of the reaction on an LB agar plate with a suitable antibiotic (e.g., 100 µg/mL ampicillin for the pTRE-Tight vector) to select the transformed bacteria. Incubate the plate overnight at 37 °C.
    3. Select individual colonies using an inoculation loop or a 20 µL tip and incubate overnight in 5 mL LB medium containing the suitable antibiotic at 37 °C with shaking. The transformed bacteria may be stored in glycerol stocks at -80 °C at a ratio of 400:600 of LB medium to glycerol.
14. Extract the plasmid DNA using silica-based plasmid columns and measure the concentration by spectrophotometry at 260 nm. Confirm the insertion of the GOI by restriction analysis and its orientation and the absence of mutations potentially introduced during RT-PCR by sequencing.

2. 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 7).
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 2.11-2.14 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.

3. Induction of the transcription inhibition of the GOI

NOTE: The cells can be pretreated with the desired treatments before doxycycline induction to assess their impact on mRNA stability (Figure 5).

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 operon²³.
   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.

4. Determining the mRNA stability of the GOI

1. Treat 1 µg of total RNA with RNAse-free DNAse I (amplification grade) to remove any trace of plasmid DNA that could interfere with subsequent DNA amplification.
   1. In a 0.2 mL PCR tube, combine 1 µg of total RNA, 1 µL of 10x DNase I reaction buffer, 1 µL of DNAse I (1 U/µL), and RNAse-free water to obtain a total volume of 10 µL.
   2. Incubate the reaction at RT for 20 min.
   3. Deactivate DNAse I by adding 1 µL of 25 mM EDTA to the 10 µL reaction mix and incubating the reaction at 70 °C for 10 min.
2. Reverse-transcribe the DNA-depleted total RNA into cDNA using a commercially available cDNA synthesis kit with a blend of oligo(dT) and random hexamer primers to improve the reverse transcription efficiency.
   1. Briefly, add 4 µL of 5x reaction mix, 1 µL of reverse transcriptase, and 4 µL of RNAse-free water to 11 µL of the DNA-depleted total RNA mix to obtain a total reaction volume of 20 µL. Mix the reaction well by pipetting it up and down.
   2. Incubate the reaction for 5 min at 25 °C, followed by 20 min at 46 °C, and then inactivate the reaction by incubating it at 95 °C for 1 min. Each reaction will yield 50 ng/µL of cDNA product.
   3. Dilute the cDNA reaction to a concentration of 5 ng/µL by adding 180 µL of molecular biology-grade water to the 20 µL reaction mix. Store the cDNA products at -80 °C or proceed immediately to performing real-time quantitative PCR (qPCR).
      NOTE: The protocol can be paused here.
3. Design forward and reverse qPCR primers specific to the GOI.
   1. Due to the endogenous expression of the GOI in the cells, the primers must be designed to amplify a 100-150 bp amplicon of the V5 epitope coupled to the GOI (Table 1).
   2. Internal reference gene primers must also be used as normalization controls. Usually, housekeeping genes, such as the beta-actin and hypoxanthine phosphoribosyltransferase 1 genes, are used as reference genes. However, these cannot be used with this induction system due to the variation of the transfection efficiency. Instead, the expression of the tTA-Ad transcript is assessed for the purposes of normalization, because its expression is constitutive in cells due to the activity of the cytomegalovirus promoter. Any variation in its expression measured by qPCR will be representative of the transfection efficiency (primers: forward 5'-GCC TGA CGA CAA GGA AAC TC-3' and reverse 5'-AGT GGG TAT GAT GCC TGT CC-3; 129 bp amplicon) and will allow the normalization of the expression of the transfected clones (Figure 3).
4. Prepare each qPCR reaction in triplicate using a SYBR Green dye master mix.
   1. Dilute the 5 ng/µL cDNA mix to a concentration of 1.25 ng/µL using molecular biology-grade water.
   2. Combine 5 µL of SYBR Green dye master mix (2x), 0.1 µL of molecular biology-grade water, 0.45 µL of 7.5 µM forward primer, 0.45 µL of 7.5 µM reverse primer, and 4 µL of 1.25 ng/µL cDNA to obtain a total reaction volume of 10 µL. Mix well by pipetting up and down in a 96 well PCR plate. Use optical adhesive film to ensure that the plate cover is sealed and to prevent contamination and evaporation.
   3. Spin down the reaction mix briefly by centrifugation and place the plate in a qPCR thermocycler.
5. Amplify the V5-tagged GOI and tTA-Ad amplicons by using the following qPCR conditions: 95 °C for 10 min as a denaturation step, followed by 40 cycles of 95 °C for 10 s, 58 °C for 15 s, and 72 °C for 20 s. A high-resolution melting curve must be generated after the amplification cycles are performed to assess the specific melting temperatures of the desired amplicons and to ensure the absence of noise amplicon peaks.
   1. Include negative controls for the qRT-PCR by performing qPCR with RNA as a template without reverse transcriptase to serve as a control for potential plasmid DNA contamination and qPCR with no cDNA added to the qPCR mix to ensure the lack of primer dimers or contaminants.
   2. The optimal cDNA concentration, primer efficiency, and concentration must be optimized according to the GOI by a standard curve assay. To do so, perform a serial dilution using cDNA from untreated cells (cultured without doxycycline). The standard curve is generated by plotting the Cq values against the log of the cDNA dilution factor, and the amplification efficiency (E) is calculated according to the slope of the standard curve using the following formula:
      E = 10^-1/slope
      NOTE: The amplification efficiency should be approximately 0.9 to 1.05. Otherwise, the primers must be redesigned.
6. Analyze the qPCR data using the comparative Cq method by normalizing the expression values of the GOI to the expression of tTA-Ad to obtain the relative expression levels of the GOI and to report its expression as a percentage of the mRNA expression of the GOI in cells from the same animal at the starting point (t = 0) (Figure 4).
7. The half-life is determined from the rate constant (K) of the GOI mRNA degradation curve using the following equation:
   t_1/2 = ln 2/K.