Name: Efficient Transcriptionally Controlled Plasmid Expression System for Investigation of the Stability of mRNA Transcripts in Primary Alveolar Epithelial Cells (Video) | JoVE Journal
Uploaded: 2020-03-06T12:30:00.000+00:00
Duration: 10 min 49 s
Description: Learn a transcriptionally controlled plasmid expression system for studying mRNA stability in primary alveolar epithelial cells. This pipette electroporation method minimizes cellular disruption.

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5 minutes

Francis Migneault was supported by a fellowship provided by the Quebec Respiratory Health Network and the Canadian Institutes of Health Research (CIHR) lung training program, a studentship from FRSQ and a studentship from the Facult&#233; des &#201;tudes Sup&#233;rieures et Postdoctorales, Universit&#233; de Montr&#233;al. This work was supported by the Gosselin-Lamarre Chair in clinical research and the Canadian Institutes of Health Research [YBMOP-79544].

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&#39;Universit&#233; de Montr&#233;al (CRCHUM).
1. Design and generation of the response plasmid expressing the gene of interest (GOI)
<ol>
	<li>Use an inducible tetracycline-off vector, such as pTRE-Tight.</li>
	<li>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.</li>
	<li>Isolate primary alveolar epithelial cells from male Sprague-Dawley rat lungs as described previously19,20.</li>
	<li>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.</li>
	<li>Reverse-transcribe the mRNA into complementary DNA (cDNA) using oligo(dT) and high-fidelity reverse transcriptase.</li>
	<li>Use high-fidelity Taq polymerase and standard overlap PCR techniques to flank the GOI with two selected restriction enzyme recognition sites using designed primers.
	<ol>
		<li>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).</li>
		<li>Have the reverse primer contain a polyadenylation signal after the stop codon.</li>
	</ol>
	</li>
	<li>Mutants can be generated by sequential deletion to study the effects of different 3&#39; UTR regions on the stability of the mRNA of the GOI using reverse primers encoding a polyadenylation site that gradually deletes the 3&#39; end of the GOI 3&#39; UTR (Figure 6). Alternatively, PCR-directed mutagenesis can be used to target a specific region of interest in the 3&#39; UTR22.</li>
	<li>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.</li>
	<li>Separate the digested vector and insert segments by electrophoresis in a 1-1.5% agarose gel (concentration depending on the size of the insert).</li>
	<li>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.</li>
	<li>Purify the collected segments from the agarose gel using a silica-based PCR purification kit and measure the concentration by spectrophotometry at 260 nm.</li>
	<li>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.</li>
	<li>Transform the ligation reaction into competent E. coli (DH5&#945;).
	<ol>
		<li>Add 1-10 ng of vector and gene to a tube containing 100 &#181;L of competent cells. Incubate the cells on ice for 30 min and then heat-shock cells at 42 &#176;C for 45 s. Place the tube on ice for 2 min and add 900 &#181;L of RT LB medium. Incubate the cells for 1 h at 37 &#176;C with shaking at 225 rpm.</li>
		<li>Spread 100 &#181;L of the reaction on an LB agar plate with a suitable antibiotic (e.g., 100 &#181;g/mL ampicillin for the pTRE-Tight vector) to select the transformed bacteria. Incubate the plate overnight at 37 &#176;C.</li>
		<li>Select individual colonies using an inoculation loop or a 20 &#181;L tip and incubate overnight in 5 mL LB medium containing the suitable antibiotic at 37 &#176;C with shaking. The transformed bacteria may be stored in glycerol stocks at -80 &#176;C at a ratio of 400:600 of LB medium to glycerol.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>
2. Transfection of the response plasmid expressing the gene of interest (GOI) into primary alveolar epithelial cells
<ol>
	<li>Isolate type II alveolar epithelial cells from rat lungs.</li>
	<li>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 &#181;g/mL trimethoprim and 17 &#181;g/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 &#176;C in 5% CO2 in a humidified incubator.</li>
	<li>On the next day, place 500 &#181;L of complete MEM without antibiotic in each well of a new 12 well plate and prewarm the plate at 37 &#176;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.</li>
	<li>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 &#181;g of GOI plasmid and 1 &#181;g of regulatory vector per well at RT. For coexpression experiments with RNA-binding proteins (RBP), 1 &#181;g of a constitutive vector (e.g., pcDNA3) expressing the RPB of interest is added to the DNA mix (Figure 7).</li>
	<li>Aspirate the medium and gently rinse the cells with PBS (without calcium and magnesium) prewarmed at 37 &#176;C.</li>
	<li>Add 5 mL of 0.05% trypsin prewarmed at 37 &#176;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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Place the tube in the electroporation device and fill it with 3.5 mL of electrolytic buffer.</li>
	<li>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.</li>
	<li>Insert the pipette in the electroporation station until there is a clicking sound.</li>
	<li>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.</li>
	<li>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 &#176;C.</li>
	<li>Repeat steps 2.11-2.14 for the remaining samples.</li>
	<li>Gently shake the plate to spread the cells evenly over the well surface. Incubate the cells at 37 &#176;C in 5% CO2 in a humidified incubator. After 2 days, replace the medium with complete MEM with antibiotics.</li>
	<li>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.</li>
</ol>
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).
<ol>
	<li>Prepare a doxycycline stock solution of 1 mg/mL in deionized water. Store the stock solution at -20 &#176;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).</li>
	<li>Prepare a fresh 1 &#181;g/mL doxycycline solution in complete MEM 72 h posttransfection and warm it to 37 &#176;C.</li>
	<li>Replace the medium with 1 mL of complete MEM containing 1 &#181;g/mL doxycycline per well to inhibit the transcription of the GOI.</li>
	<li>Incubate multiple wells at 37 &#176;C in 5% CO2 for different amounts of time from 15 min-6 h to assess the mRNA half-life of the GOI.</li>
	<li>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 &#181;L of buffer per well and shaking the plate to homogenize the cells.</li>
	<li>Isolate the RNA according to the manufacturer&#39;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.</li>
</ol>
4. Determining the mRNA stability of the GOI
<ol>
	<li>Treat 1 &#181;g of total RNA with RNAse-free DNAse I (amplification grade) to remove any trace of plasmid DNA that could interfere with subsequent DNA amplification.
	<ol>
		<li>In a 0.2 mL PCR tube, combine 1 &#181;g of total RNA, 1 &#181;L of 10x DNase I reaction buffer, 1 &#181;L of DNAse I (1 U/&#181;L), and RNAse-free water to obtain a total volume of 10 &#181;L.</li>
		<li>Incubate the reaction at RT for 20 min.</li>
		<li>Deactivate DNAse I by adding 1 &#181;L of 25 mM EDTA to the 10 &#181;L reaction mix and incubating the reaction at 70 &#176;C for 10 min.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Briefly, add 4 &#181;L of 5x reaction mix, 1 &#181;L of reverse transcriptase, and 4 &#181;L of RNAse-free water to 11 &#181;L of the DNA-depleted total RNA mix to obtain a total reaction volume of 20 &#181;L. Mix the reaction well by pipetting it up and down.</li>
		<li>Incubate the reaction for 5 min at 25 &#176;C, followed by 20 min at 46 &#176;C, and then inactivate the reaction by incubating it at 95 &#176;C for 1 min. Each reaction will yield 50 ng/&#181;L of cDNA product.</li>
		<li>Dilute the cDNA reaction to a concentration of 5 ng/&#181;L by adding 180 &#181;L of molecular biology-grade water to the 20 &#181;L reaction mix. Store the cDNA products at -80 &#176;C or proceed immediately to performing real-time quantitative PCR (qPCR). 
		NOTE: The protocol can be paused here.</li>
	</ol>
	</li>
	<li>Design forward and reverse qPCR primers specific to the GOI.
	<ol>
		<li>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).</li>
		<li>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&#39;-GCC TGA CGA CAA GGA AAC TC-3&#39; and reverse 5&#39;-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).</li>
	</ol>
	</li>
	<li>Prepare each qPCR reaction in triplicate using a SYBR Green dye master mix.
	<ol>
		<li>Dilute the 5 ng/&#181;L cDNA mix to a concentration of 1.25 ng/&#181;L using molecular biology-grade water.</li>
		<li>Combine 5 &#181;L of SYBR Green dye master mix (2x), 0.1 &#181;L of molecular biology-grade water, 0.45 &#181;L of 7.5 &#181;M forward primer, 0.45 &#181;L of 7.5 &#181;M reverse primer, and 4 &#181;L of 1.25 ng/&#181;L cDNA to obtain a total reaction volume of 10 &#181;L. 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.</li>
		<li>Spin down the reaction mix briefly by centrifugation and place the plate in a qPCR thermocycler.</li>
	</ol>
	</li>
	<li>Amplify the V5-tagged GOI and tTA-Ad amplicons by using the following qPCR conditions: 95 &#176;C for 10 min as a denaturation step, followed by 40 cycles of 95 &#176;C for 10 s, 58 &#176;C for 15 s, and 72 &#176;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.
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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).</li>
	<li>The half-life is determined from the rate constant (K) of the GOI mRNA degradation curve using the following equation: 
	t1/2 = ln 2/K.</li>
</ol>

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<li>Migneault, F. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Modulation+de+la+stabilit%C3%A9+de+l%27ARNm+alphaENaC+dans+les+cellules+%C3%A9pith%C3%A9liales+alv%C3%A9olaires%3A+d%C3%A9termination+du+r%C3%B4le+des+s%C3%A9quences+3%27+non+traduites%22">Modulation de la stabilité de l'ARNm alphaENaC dans les cellules épithéliales alvéolaires: détermination du rôle des séquences 3' non traduites</a>. , Université de Montréal. (2015).</li>
<li>Grzesik, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+gene+delivery+to+primary+alveolar+epithelial+cells+by+nucleofection%5BTitle%5D)+AND+%22American+Journal+of+Physiology-Lung+Cellular+and+Molecular+Physiology%22%5BJournal%5D)">Efficient gene delivery to primary alveolar epithelial cells by nucleofection.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 305 (11), L786-L794 (2013).</li>
<li>Sourdeval, M., Lemaire, C., Brenner, C., Boisvieux-Ulrich, E., Marano, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+doxycycline-induced+cytotoxicity+on+human+bronchial+epithelial+cells%5BTitle%5D)+AND+%22Frontiers+in+Bioscience%22%5BJournal%5D)">Mechanisms of doxycycline-induced cytotoxicity on human bronchial epithelial cells.</a> Frontiers in Bioscience. 11, 3036-3048 (2006).</li>
<li>Moon, A., Gil, S., Gill, S. E., Chen, P., Matute-Bello, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Doxycycline+impairs+neutrophil+migration+to+the+airspaces+of+the+lung+in+mice+exposed+to+intratracheal+lipopolysaccharide%5BTitle%5D)+AND+%22Journal+of+Inflammation-London%22%5BJournal%5D)">Doxycycline impairs neutrophil migration to the airspaces of the lung in mice exposed to intratracheal lipopolysaccharide.</a> Journal of Inflammation-London. 9 (1), 31(2012).</li>
<li>Hovel, H., Frieling, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+use+of+doxycycline%2C+mezlocillin+and+clotrimazole+in+cell+culture+media+as+contamination+prophylaxis%5BTitle%5D)+AND+%22Developments+in+Biological+Standardization%22%5BJournal%5D)">The use of doxycycline, mezlocillin and clotrimazole in cell culture media as contamination prophylaxis.</a> Developments in Biological Standardization. 66, 23-28 (1987).</li>
<li>Houseley, J., Tollervey, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+many+pathways+of+RNA+degradation%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">The many pathways of RNA degradation.</a> Cell. 136 (4), 763-776 (2009).</li>
<li>Tani, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+determination+of+RNA+stability+reveals+hundreds+of+short-lived+noncoding+transcripts+in+mammals%5BTitle%5D)+AND+%22Genome+Research%22%5BJournal%5D)">Genome-wide determination of RNA stability reveals hundreds of short-lived noncoding transcripts in mammals.</a> Genome Research. 22 (5), 947-956 (2012).</li>
</ol>

The authors have no conflicts of interest to disclose.

The low transfection rate of alveolar epithelial cells in primary culture has been a serious limitation for the use of the Tet-Off system to assess mRNA stability in these cells. However, this limitation was overcome by pipette electroporation, allowing a 25-30% transfection efficiency (Figure 1 and Figure 3)26.
The measurement of transcript stability is fundamental to understanding the modulation of a given mR...

Several techniques have been developed to determine the half-life of mRNAs. The pulse-chase decay technique, which utilizes labeled mRNAs, allows for the simultaneous evaluation of a large pool of mRNAs with minimal cellular disturbance. However, this approach does not allow a direct estimation of the half-life of a single gene transcript and cannot be implemented to study the posttranscriptional modulation of an mRNA following stimulation with growth factors, ROS, alarmins, or inflammation1.
The use of transcription inhibitors, such as actinomycin D and &#945;-amanitin, is a relatively simple method for measuring mRNA degradation kinetics over time. One main advantage of this approach over that of previous techniques, (i.e., pulse-chase) relies on the ability to directly estimate the half-life of a given transcript and compare how different treatments could affect its degradation kinetics. However, the significant deleterious impact of transcription inhibitors on cell physiology represents a major drawback of the approach2. Indeed, the inhibition of the whole cell transcriptome with these drugs has the negative side effect of perturbing the synthesis of key elements involved in mRNA stability, such as microRNAs (miRNAs), as well as the expression and synthesis of RNA-binding proteins, which are important for mRNA degradation and stability. The severe perturbation of gene transcription by these drugs could therefore artefactually modify the degradation curves of transcripts.
The promoter induction system represents a third approach to measure the half-life of a specific mRNA. This method measures the degradation of a specific mRNA in a similar way as methods that use transcription inhibitors. Two types of induction systems are frequently used: the serum-induced c-fos promoter3 and the Tet-Off inducible system4. With the c-fos system, the use of transcription inhibitors that can be toxic to the cell is not needed. However, this method requires cell cycle synchronization, which prevents the evaluation of the actual stability of a transcript during interphase5. In contrast, the Tet-Off system allows the strong expression of the gene of interest (GOI) under the control of a synthetic tetracycline-regulated promoter. This system requires the presence of two elements that must be cotransfected into the cell to be functional. The first plasmid (pTet-Off) expresses the regulatory protein tTA-Adv, a hybrid synthetic transcription factor composed of the prokaryotic repressor TetR (from Escherichia coli) fused to three transcription transactivation domains from the viral protein HSV VP16. The GOI is cloned into the pTRE-Tight plasmid under the control of a synthetic promoter (PTight), comprising the minimal sequence of the cytomegalovirus (CMV) promoter fused to seven repeats of the tetO operator sequence. The transcription of the gene downstream of PTight is dependent on the interaction of TetR with tetO. In the presence of tetracycline or its derivative, doxycycline, the TetR repressor loses its affinity for the tetO operator, leading to a cessation of transcription4. The characteristics of the Tet-Off system make it an ideal model for the study of specific mRNA expression in eukaryotic cells while avoiding potential pleiotropic effects that are secondary to the absence of prokaryotic regulatory sequences in eukaryotic cell6. Usually, doubly stable Tet-Off cell lines (HEK 293, HeLa, and PC12) are used with this system to integrate copies of the regulator and response plasmids for convenient access to controllable gene expression7,8,9.
Several models of alveolar epithelial cells in culture have been used to study the cellular and molecular biology of the alveolar epithelium. For years, researchers have extensively utilized human or rodent primary cells10,11 as well as immortalized cell lines such as human A549 or rat RLE-6TN cells12,13. Although they are generally less proliferative and more difficult to culture and to transfect, alveolar epithelial cells in primary culture remain the gold standard for the study of the function and dysfunction of the alveolar epithelium in physiological and pathological conditions. Indeed, immortalized cell lines such as A549 cells do not exhibit the complex characteristics and phenotypes of primary cells, whereas alveolar epithelial cells in primary culture recapitulate the main properties of the alveolar epithelium, in particular the ability to form a polarized and tight barrier14,15. Unfortunately, these cells are very resistant to conventional transfection techniques, such as those utilizing liposomes, making the use of a promoter-induced system such as Tet-Off very difficult.
The posttranscriptional modulation of mRNAs is one of the most effective methods for rapidly modulating the gene expression of a transcript16. The mRNA 3&#39; untranslated region (3&#39; UTR) plays an important role in this mechanism. It has been shown that, unlike the 5&#39; UTR, there is an exponential correlation between the length of the 3&#39; UTR and the cellular and morphological complexities of an organism. This correlation suggests that the 3&#39; UTR, like the mRNA coding regions, has been subjected to natural selection to allow for increasingly complex posttranscriptional modulation throughout evolution17. The 3&#39; UTR contains several binding sites for proteins and miRNAs that affect the stability and translation of the transcript.
In the present work, we developed a tool to investigate the role of highly conserved domains in the 3&#39; UTR of a GOI for the control of transcript stability. We focused on the epithelial sodium channel, alpha subunit (&#945;ENaC), which plays a key role in alveolar epithelial physiology18. Alveolar epithelial cells in primary culture were successfully transiently transfected with the two components of the Tet-Off system, which allows for the study of the role of the 3&#39; UTR in mRNA stability with a system that minimally affects cell physiology and metabolism in comparison to the use of transcription inhibitors with other protocols. A cloning strategy was developed to differentiate the expression of the GOI from that of the endogenous gene using a nonendogenously expressed epitope (V5). The response and regulatory plasmids were then transferred into alveolar epithelial cells using a pipette electroporation technique. Subsequently, the expression of the transcript was measured by incubating the cells with doxycycline at different time intervals. The half-life of the transcript was evaluated by RT-qPCR with a modified Cq method using the transfected tTA-Ad mRNA product for normalization. Through our protocol, we offer a convenient way for studying the posttranscriptional modulation of a transcript under different conditions and defining the involvement of the untranslated regions in more detail.

<table><tbody><tr><td>Actinomycin D</td><td>Sigma-Aldrich</td><td>A9415</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma-Aldrich</td><td>A1593</td><td></td></tr><tr><td>Bright-LineHemacytometer</td><td>Sigma-Aldrich</td><td>Z359629</td><td></td></tr><tr><td>Chloroform - Molecular biology grade</td><td>Sigma-Aldrich</td><td>C2432</td><td></td></tr><tr><td>ClaI</td><td>New England Biolabs</td><td>R0197S</td><td></td></tr><tr><td>Cycloheximide</td><td>Sigma-Aldrich</td><td>C7698</td><td></td></tr><tr><td>DM IL LED Inverted Microscope with Phase Contrast</td><td>Leica</td><td>-</td><td></td></tr><tr><td>DNase I, Amplification Grade</td><td>Invitrogen</td><td>18068015</td><td></td></tr><tr><td>Doxycycline hyclate</td><td>Sigma-Aldrich</td><td>D9891-1G</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate-buffered Saline (D-PBS), without calcium and magnesium</td><td>Wisent Bioproducts</td><td>311-425-CL</td><td></td></tr><tr><td>Ethanol - Molecular biology grade</td><td>Fisher Scientific</td><td>BP2818100</td><td></td></tr><tr><td>Excella E25 ConsoleIncubatorShaker</td><td>Eppendorf</td><td>1220G76</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>HEPES pH 7.3</td><td>Sigma-Aldrich</td><td>H3784</td><td></td></tr><tr><td>Heracell 240i</td><td>ThermoFisher Scientific</td><td>51026420</td><td></td></tr><tr><td>iScript cDNA Synthesis Kit</td><td>Bio-Rad Laboratories</td><td>1708890</td><td></td></tr><tr><td>Isopropanol - Molecular biology grade</td><td>Sigma-Aldrich</td><td>I9516</td><td></td></tr><tr><td>LB Broth (Lennox)</td><td>Sigma-Aldrich</td><td>L3022</td><td></td></tr><tr><td>LB Broth with agar (Lennox)</td><td>Sigma-Aldrich</td><td>L2897</td><td></td></tr><tr><td>L-glutamine</td><td>Sigma-Aldrich</td><td>G7513</td><td></td></tr><tr><td>Lipopolysaccharides fromPseudomonas aeruginosa10</td><td>Sigma-Aldrich</td><td>L9143</td><td></td></tr><tr><td>MEM, powder</td><td>Gibco</td><td>61100103</td><td></td></tr><tr><td>MicroAmp Optical 96-Well Reaction Plate</td><td>Applied Biosystems</td><td>N8010560</td><td></td></tr><tr><td>MicroAmp Optical Adhesive Film</td><td>Applied Biosystems</td><td>4360954</td><td></td></tr><tr><td>MSC-Advantage Class II Biological Safety Cabinets</td><td>ThermoFisher Scientific</td><td>51025413</td><td></td></tr><tr><td>Mupid-exU electrophoresis system</td><td>Takara Bio</td><td>AD140</td><td></td></tr><tr><td>NanoDrop 2000c</td><td>ThermoFisher Scientific</td><td>ND-2000</td><td></td></tr><tr><td>Neon Transfection System 10 &amp;micro;L Kit</td><td>Invitrogen</td><td>MPK1025</td><td></td></tr><tr><td>Neon Transfection System Starter Pack</td><td>Invitrogen</td><td>MPK5000S</td><td></td></tr><tr><td>NheI</td><td>New England Biolabs</td><td>R0131S</td><td></td></tr><tr><td>One Shot OmniMAX 2 T1RChemically CompetentE. coli</td><td>Invitrogen</td><td>C854003</td><td></td></tr><tr><td>pcDNA3 vector</td><td>ThermoFisher Scientific</td><td>V790-20</td><td></td></tr><tr><td>pcDNA3-EGFP plasmid</td><td>Addgene</td><td>13031</td><td></td></tr><tr><td>PlatinumTaqDNA Polymerase High Fidelity</td><td>Invitrogen</td><td>11304011</td><td></td></tr><tr><td>pTet-Off Advanced vector</td><td>Takara Bio</td><td>631070</td><td></td></tr><tr><td>pTRE-Tight vector</td><td>Takara Bio</td><td>631059</td><td></td></tr><tr><td>Purified alveolar epithelial cells</td><td>n.a.</td><td>n.a.</td><td></td></tr><tr><td>QIAEX II Gel Extraction Kit</td><td>QIAGEN</td><td>20021</td><td></td></tr><tr><td>QIAGEN Plasmid Maxi Kit</td><td>QIAGEN</td><td>12162</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>QIAGEN</td><td>27104</td><td></td></tr><tr><td>QuantStudio 6 and 7 Flex Real-Time PCR System Software</td><td>Applied Biosystems</td><td>n.a.</td><td></td></tr><tr><td>QuantStudio 6 Flex Real-Time PCR System, 96-well Fast</td><td>Applied Biosystems</td><td>4485697</td><td></td></tr><tr><td>Recombinant Rat TNF-alpha Protein</td><td>R&amp;amp;D Systems</td><td>510-RT-010</td><td></td></tr><tr><td>Septra</td><td>Sigma-Aldrich</td><td>A2487</td><td></td></tr><tr><td>Shrimp Alkaline Phosphatase (rSAP)</td><td>New England Biolabs</td><td>M0371S</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S5761</td><td></td></tr><tr><td>SsoAdvanced Universal SYBR Green Supermix</td><td>Bio-Rad Laboratories</td><td>1725270</td><td></td></tr><tr><td>SuperScript IV Reverse Transcriptase</td><td>Invitrogen</td><td>18090010</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>ThermoFisher Scientific</td><td>EL0011</td><td></td></tr><tr><td>Tet System Approved FBS</td><td>Takara Bio</td><td>631367</td><td></td></tr><tr><td>Tobramycin</td><td>Sigma-Aldrich</td><td>T4014</td><td></td></tr><tr><td>TRIzol Reagent</td><td>Invitrogen</td><td>15596018</td><td></td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>Gibco</td><td>25300054</td><td></td></tr><tr><td>UltraPure Agarose</td><td>Invitrogen</td><td>16500500</td><td></td></tr><tr><td>Water, Molecular biology Grade</td><td>Wisent Bioproducts</td><td>809-115-EL</td><td></td></tr></tbody></table>

efficient transcriptionally controlled plasmid expression system for investigation of the stability of mrna transcripts in primary alveolar epithelial cells

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

Studying posttranscriptional regulation is fundamental to understanding the modulation of a given messenger RNA (mRNA) and its impact on cell homeostasis and metabolism. Indeed, fluctuations in transcript expression could modify the translation efficiency and ultimately the cellular activity of a transcript. Several experimental approaches have been developed to investigate the half-life of mRNA although some of these methods have limitations that prevent the proper study of posttranscriptional modulation. A promoter induction system can express a gene of interest under the control of a synthetic tetracycline-regulated promoter. This method allows the half-life estimation of a given mRNA under any experimental condition without disturbing cell homeostasis. One major drawback of this method is the necessity to transfect cells, which limits the use of this technique in isolated primary cells that are highly resistant to conventional transfection techniques. Alveolar epithelial cells in primary culture have been used extensively to study the cellular and molecular biology of the alveolar epithelium. The unique characteristics and phenotype of primary alveolar cells make it essential to study the posttranscriptional modulations of genes of interest in these cells. Therefore, our aim was to develop a novel tool to investigate the posttranscriptional modulations of mRNAs of interest in alveolar epithelial cells in primary culture. We designed a fast and efficient transient transfection protocol to insert a transcriptionally controlled plasmid expression system into primary alveolar epithelial cells. This cloning strategy, using a viral epitope to tag the construct, allows for the easy discrimination of construct expression from that of endogenous mRNAs. Using a modified &#916;&#916; quantification cycle (Cq) method, the expression of the transcript can then be quantified at different time intervals to measure its half-life. Our data demonstrate the efficiency of this novel approach in studying posttranscriptional regulation in various pathophysiological conditions in primary alveolar epithelial cells.

This protocol was successfully used to generate a Tet-Off transcriptionally controlled plasmid expression system to evaluate the importance of different portions of the αENaC 3' UTR in the modulation of transcript stability in primary alveolar epithelial cells.
The first step in the implementation of this system was to establish a fast, easy, and efficient transfection technique for alveolar epithelial cells in primary culture, ...

Here, we present a tool that can be used to study the posttranscriptional modulation of a transcript in primary alveolar epithelial cells by using an inducible expression system coupled to a pipette electroporation technique.

Studying posttranscriptional regulation is fundamental to understanding the modulation of a given messenger RNA (mRNA) and its impact on cell homeostasis ...

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Research

JoVE Journal

Genetics

RNA polymerase II transcriptional kinetics are measured on specific genes in living cells. mRNAs transcribed from the gene of interest are fluorescently tagged and using Fluorescence Recovery After Photobleaching (FRAP) the in vivo kinetics of transcriptional elongation are obtained.

Measuring the Kinetics of mRNA Transcription in Single Living Cells

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.

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

Transgenic mice or viral vectors have been used to increase protein expression within the lung. However, these techniques are time-consuming, technically challenging and have off-target effects that can confound results. Our protein transfection protocol uses a lipid based transfection reagent and an ultrafine microsprayer to uniformly deliver active protein to lung cells.

Protein Transfection of Mouse Lung

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3’ UTR.

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

Controllable Ion Channel Expression through Inducible Transient Transfection

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

We developed an intronic microRNA biogenesis reporter assay to be used in cells in vitro with four plasmids: one with intronic miRNA, one with the target, one to overexpress a regulatory protein, and one for Renilla luciferase. The miRNA was processed and could control luciferase expression by binding to the target sequence.

A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells

Presented here is a reproducible, affordable, and robust method for the isolation and expansion of primary bronchial epithelial cells for long-term biobanking and the generation of differentiated epithelial cells by culture at the air-liquid interface.

Isolating Bronchial Epithelial Cells from Resected Lung Tissue for Biobanking and Establishing Well-Differentiated Air-Liquid Interface Cultures

This protocol presents a flow cytometry-based method to isolate viable alveolar type II epithelial cells (AECII) from neonatal, juvenile, or adult murine lungs (infected or non-infected). This approach enables downstream molecular and/or functional studies of AECII to identify their roles during lung inflammation and infection.

Isolation of Alveolar Type II Epithelial Cells from Neonatal, Juvenile, and Adult Murine Lungs Adaptable to Infectious Experimental Settings

Learn a transcriptionally controlled plasmid expression system for studying mRNA stability in primary alveolar epithelial cells. This pipette electroporation method minimizes cellular disruption.

Efficient Transcriptionally Controlled Plasmid Expression System for Investigation of the Stability of mRNA Transcripts in Primary Alveolar Epithelial Cells (Video) | JoVE Journal

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

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