Efficient Transcriptionally Controlled Plasmid Expression System for Investigation of the Stability of mRNA Transcripts in Primary Alveolar Epithelial Cells
| Autor: | Frédéric Gagnon, Yves Berthiaume, Francis Migneault, Emmanuelle Brochiero, André Dagenais |
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| Rok vydání: | 2020 |
| Předmět: |
Transcription
Genetic RNA Stability General Chemical Engineering Cell Transfection General Biochemistry Genetics and Molecular Biology Alveolar cells Transcription (biology) medicine Animals RNA Messenger Epithelial Sodium Channels 3' Untranslated Regions Cells Cultured DNA Primers Inflammation Messenger RNA General Immunology and Microbiology Chemistry Three prime untranslated region Electroporation General Neuroscience RNA-Binding Proteins Translation (biology) Rats Cell biology Kinetics medicine.anatomical_structure Gene Expression Regulation Alveolar Epithelial Cells Doxycycline Dactinomycin Plasmids |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| DOI: | 10.3791/60654-v |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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