A system for creating stable cell lines that express a gene of interest from a bidirectional and regulatable herpes simplex virus type 1 promoter

Expression systems used to study the biological function of a gene of interest can have limited utility due to three major factors: i) weak or heterogeneous gene expression; ii) poorly controlled gene expression; and iii) low efficiencies of stable integration and persistent expression. We envisione...

Full description

Saved in:
Bibliographic Details
Published in:PloS one 2015-03, Vol.10 (3), p.e0122253
Main Authors: Chambers, Christopher B, Halford, William P, Geltz, Joshua, Villamizar, Olga, Gross, Jeffrey, Embalabala, Alison, Gershburg, Edward, Wilber, Andrew
Format: Article
Language:English
Subjects:
Antibiotics
Biology
Biotechnology
Cell Line
Cell Line, Tumor
Cell lines
Chromosomes
Cloning
DNA Transposable Elements
Doxycycline
Doxycycline - pharmacology
Drug resistance
Gene expression
Gene Expression - drug effects
Gene Expression - genetics
Gene Expression Regulation, Viral - drug effects
Gene Expression Regulation, Viral - genetics
Genes
Genetic engineering
Genomes
HEK293 Cells
HeLa Cells
Herpes simplex
Herpes simplex virus
Herpes viruses
Herpesvirus 1, Human - genetics
Humans
Immunology
Integration
Medicine
Plasmids
Promoter Regions, Genetic - drug effects
Promoter Regions, Genetic - genetics
Proteins
Tetracycline - pharmacology
Trans-Activators - pharmacology
Transcription
Transcription, Genetic - drug effects
Transcription, Genetic - genetics
Viruses
VP16 protein
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Expression systems used to study the biological function of a gene of interest can have limited utility due to three major factors: i) weak or heterogeneous gene expression; ii) poorly controlled gene expression; and iii) low efficiencies of stable integration and persistent expression. We envisioned that the ideal system should be tightly controlled and coupled with the ability to efficiently create and identify stable cell lines. Herein, we describe a system based upon a bidirectional Herpes simplex virus type 1 promoter that is naturally responsive to the VP16 transactivator and modified to permit tetracycline-regulated transcription on one side while maintaining constitutive activity on the other side. Incorporation of this element into the Sleeping Beauty transposon resulted in a novel bidirectional system with the capacity for high-efficiency stable integration. Using this system, we created stable cell lines in which expression of a gene of interest was tightly and uniformly controlled across a broad range of levels via a novel combination of doxycycline-sensitive de-repression and VP16-mediated sequence-specific induction. The unique characteristics of this system address major limitations of current methods and provide an excellent strategy to investigate the effects of gene dosing in mammalian models.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0122253