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 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…

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