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 mR....

Protocol

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 epithelia....

Results

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, .......

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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