Inducible Tet-Off Regulatable System: An In Vitro Method to Modulate Gene Expression in Cultured Cells Using Tetracycline-Off System
Published: April 30, 2023
Abstract
Source: Migneault, F. et al, Efficient Transcriptionally Controlled Plasmid Expression System for Investigation of the Stability of mRNA Transcripts in Primary Alveolar Epithelial Cells. J. Vis. Exp. (2020).
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.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Transfection of the response plasmid expressing the gene of interest (GOI) into primary alveolar epithelial cells Isolate type II alveolar epithelial cells from rat lungs. Seed the cells at a density of 1 x 106 cells/cm2 in 100 mm Petri dishes with complete mini…
Representative Results
Figure 1: Efficiency of the transfection of alveolar epithelial cells in primary culture by pipette electroporation. Primary alveolar epithelial cells were transiently transfected with 2 µg of a pcDNA3 plasmid (empty, clones #1 and #2) that expressed or did not express GFP protein. Transfection efficiency was assessed 48 h following transfection by (A) fluorescence micr…
Divulgaciones
The authors have nothing to disclose.
Materials
| Bright-LineHemacytometer | Sigma-Aldrich | Z359629 | |
| DM IL LED Inverted Microscope with Phase Contrast | Leica | ||
| Dulbecco’s Phosphate-buffered Saline (D-PBS), without calcium and magnesium | Wisent Bioproducts | 311-425-CL | |
| Doxycycline hyclate | Sigma-Aldrich | D9891-1G | |
| MEM, powder | Gibco | 61100103 | |
| MSC-Advantage Class II Biological Safety Cabinets | ThermoFisher Scientific | 51025413 | |
| Neon Transfection System 10 µL Kit | Invitrogen | MPK1025 | |
| Neon Transfection System Starter Pack | Invitrogen | MPK5000S | |
| pcDNA3 vector | ThermoFisher Scientific | V790-20 | |
| pcDNA3-EGFP plasmid | Addgene | 13031 | |
| 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 RealTime PCR System Software | Applied Biosystems | n.a. | |
| Tet System Approved FBS | Takara Bio | 631367 |
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Citar este artículo
Inducible Tet-Off Regulatable System: An In Vitro Method to Modulate Gene Expression in Cultured Cells Using Tetracycline-Off System. J. Vis. Exp. (Pending Publication), e20979, doi: (2023).