Inducible and reversible lentiviral and recombination mediated cassette exchange (RMCE) systems for controlling gene expression

Bersten, David C., Sullivan, Adrienne E., Li, Dian, Bhakti, Veronica, Bent, Stephen J. and Whitelaw, Murray L. (2015) Inducible and reversible lentiviral and recombination mediated cassette exchange (RMCE) systems for controlling gene expression. PLoS One, 10 3: . doi:10.1371/journal.pone.0116373


Author Bersten, David C.
Sullivan, Adrienne E.
Li, Dian
Bhakti, Veronica
Bent, Stephen J.
Whitelaw, Murray L.
Title Inducible and reversible lentiviral and recombination mediated cassette exchange (RMCE) systems for controlling gene expression
Journal name PLoS One   Check publisher's open access policy
ISSN 1932-6203
Publication date 2015-03-13
Sub-type Article (original research)
DOI 10.1371/journal.pone.0116373
Open Access Status DOI
Volume 10
Issue 3
Total pages 20
Place of publication San Francisco, United States
Publisher Public Library of Science
Collection year 2016
Language eng
Formatted 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.
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ
Additional Notes Article number e0116373

Document type: Journal Article
Sub-type: Article (original research)
Collections: Non HERDC
Institute for Molecular Bioscience - Publications
 
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Created: Tue, 31 May 2016, 11:15:12 EST by Stephen Bent on behalf of Learning and Research Services (UQ Library)