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

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

1. Isolate type II alveolar epithelial cells from rat lungs.
2. 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 µg/mL trimethoprim and 17 µg/mL su....

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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