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Type: Journal article
Title: Inducible and reversible lentiviral and recombination mediated cassette exchange (RMCE) systems for controlling gene expression
Author: Bersten, D.
Sullivan, A.
Li, D.
Bhakti, V.
Bent, S.
Whitelaw, M.
Citation: PLoS One, 2015; 10(3):e0116373-1-e0116373-20
Publisher: Public Library of Science
Issue Date: 2015
ISSN: 1932-6203
Statement of
David C. Bersten, Adrienne E. Sullivan, Dian Li, Veronica Bhakti, Stephen J. Bent, Murray L. Whitelaw
Abstract: Manipulation of gene expression to invoke loss of function (LoF) or gain of function (GoF) phenotypes is important for interrogating complex biological questions both in vitro and in vivo. Doxycycline (Dox)-inducible gene expression systems are commonly used although success is often limited by high background and insufficient sensitivity to Dox. Here we develop broadly applicable platforms for reliable, tightly controlled and reversible Dox-inducible systems for lentiviral mediated generation of cell lines or FLP Recombination-Mediated Cassette Exchange (RMCE) into the Collagen 1a1 (Col1a1) locus (FLP-In Col1a1) in mouse embryonic stem cells. We significantly improve the flexibility, usefulness and robustness of the Dox-inducible system by using Tetracycline (Tet) activator (Tet-On) variants which are more sensitive to Dox, have no background activity and are expressed from single Gateway-compatible constructs. We demonstrate the usefulness of these platforms in ectopic gene expression or gene knockdown in multiple cell lines, primary neurons and in FLP-In Col1a1 mouse embryonic stem cells. We also improve the flexibility of RMCE Dox-inducible systems by generating constructs that allow for tissue or cell type-specific Dox-inducible expression and generate a shRNA selection algorithm that can effectively predict potent shRNA sequences able to knockdown gene expression from single integrant constructs. These platforms provide flexible, reliable and broadly applicable inducible expression systems for studying gene function.
Keywords: Cell Line; Cell Line, Tumor; Animals; Humans; Mice; Lentivirus; Doxycycline; Collagen Type I; RNA, Small Interfering; DNA, Complementary; Gene Transfer Techniques; Mutagenesis, Insertional; Gene Expression Regulation; Recombination, Genetic; Gene Knockdown Techniques; HEK293 Cells
Rights: © 2015 Bersten et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
RMID: 0030025233
DOI: 10.1371/journal.pone.0116373
Appears in Collections:Molecular and Biomedical Science publications

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