Efficient Transcriptionally Controlled Plasmid Expression System for Investigation of the Stability of mRNA Transcripts in Primary Alveolar Epithelial Cells
Summary
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.
Abstract
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 ΔΔ 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.
Introduction
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 α-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' untranslated region (3' UTR) plays an important role in this mechanism. It has been shown that, unlike the 5' UTR, there is an exponential correlation between the length of the 3' UTR and the cellular and morphological complexities of an organism. This correlation suggests that the 3' UTR, like the mRNA coding regions, has been subjected to natural selection to allow for increasingly complex posttranscriptional modulation throughout evolution17. The 3' 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' UTR of a GOI for the control of transcript stability. We focused on the epithelial sodium channel, alpha subunit (αENaC), 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' 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.
Protocol
Representative Results
Discussion
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…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
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 Faculté des Études Supérieures et Postdoctorales, Université de Montréal. This work was supported by the Gosselin-Lamarre Chair in clinical research and the Canadian Institutes of Health Research [YBMOP-79544].
Materials
| Actinomycin D | Sigma-Aldrich | A9415 | |
| Ampicillin | Sigma-Aldrich | A1593 | |
| Bright-LineHemacytometer | Sigma-Aldrich | Z359629 | |
| Chloroform – Molecular biology grade | Sigma-Aldrich | C2432 | |
| ClaI | New England Biolabs | R0197S | |
| Cycloheximide | Sigma-Aldrich | C7698 | |
| DM IL LED Inverted Microscope with Phase Contrast | Leica | – | |
| DNase I, Amplification Grade | Invitrogen | 18068015 | |
| Doxycycline hyclate | Sigma-Aldrich | D9891-1G | |
| Dulbecco’s Phosphate-buffered Saline (D-PBS), without calcium and magnesium | Wisent Bioproducts | 311-425-CL | |
| Ethanol – Molecular biology grade | Fisher Scientific | BP2818100 | |
| Excella E25 ConsoleIncubatorShaker | Eppendorf | 1220G76 | |
| Glycerol | Sigma-Aldrich | G5516 | |
| HEPES pH 7.3 | Sigma-Aldrich | H3784 | |
| Heracell 240i | ThermoFisher Scientific | 51026420 | |
| iScript cDNA Synthesis Kit | Bio-Rad Laboratories | 1708890 | |
| Isopropanol – Molecular biology grade | Sigma-Aldrich | I9516 | |
| LB Broth (Lennox) | Sigma-Aldrich | L3022 | |
| LB Broth with agar (Lennox) | Sigma-Aldrich | L2897 | |
| L-glutamine | Sigma-Aldrich | G7513 | |
| Lipopolysaccharides fromPseudomonas aeruginosa10 | Sigma-Aldrich | L9143 | |
| MEM, powder | Gibco | 61100103 | |
| MicroAmp Optical 96-Well Reaction Plate | Applied Biosystems | N8010560 | |
| MicroAmp Optical Adhesive Film | Applied Biosystems | 4360954 | |
| MSC-Advantage Class II Biological Safety Cabinets | ThermoFisher Scientific | 51025413 | |
| Mupid-exU electrophoresis system | Takara Bio | AD140 | |
| NanoDrop 2000c | ThermoFisher Scientific | ND-2000 | |
| Neon Transfection System 10 µL Kit | Invitrogen | MPK1025 | |
| Neon Transfection System Starter Pack | Invitrogen | MPK5000S | |
| NheI | New England Biolabs | R0131S | |
| One Shot OmniMAX 2 T1RChemically CompetentE. coli | Invitrogen | C854003 | |
| pcDNA3 vector | ThermoFisher Scientific | V790-20 | |
| pcDNA3-EGFP plasmid | Addgene | 13031 | |
| PlatinumTaqDNA Polymerase High Fidelity | Invitrogen | 11304011 | |
| 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 Real-Time PCR System Software | Applied Biosystems | n.a. | |
| QuantStudio 6 Flex Real-Time PCR System, 96-well Fast | Applied Biosystems | 4485697 | |
| Recombinant Rat TNF-alpha Protein | R&D Systems | 510-RT-010 | |
| Septra | Sigma-Aldrich | A2487 | |
| Shrimp Alkaline Phosphatase (rSAP) | New England Biolabs | M0371S | |
| Sodium bicarbonate | Sigma-Aldrich | S5761 | |
| SsoAdvanced Universal SYBR Green Supermix | Bio-Rad Laboratories | 1725270 | |
| SuperScript IV Reverse Transcriptase | Invitrogen | 18090010 | |
| T4 DNA Ligase | ThermoFisher Scientific | EL0011 | |
| Tet System Approved FBS | Takara Bio | 631367 | |
| Tobramycin | Sigma-Aldrich | T4014 | |
| TRIzol Reagent | Invitrogen | 15596018 | |
| Trypsin-EDTA (0.05%), phenol red | Gibco | 25300054 | |
| UltraPure Agarose | Invitrogen | 16500500 | |
| Water, Molecular biology Grade | Wisent Bioproducts | 809-115-EL |
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