Inducible and reversible lentiviral and Recombination Mediated Cassette Exchange (RMCE) systems for controlling gene expression

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...

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Bibliographic Details
Published in:PloS one 2015-03, Vol.10 (3), p.e0116373-e0116373
Main Authors: Bersten, David C, Sullivan, Adrienne E, Li, Dian, Bhakti, Veronica, Bent, Stephen J, Whitelaw, Murray L
Format: Article
Language:English
Subjects:
Animals
Artificial chromosomes
Biochemistry
Biotechnology
Cell Line
Cell Line, Tumor
Cell lines
Collagen
Collagen (type I)
Collagen Type I - genetics
DNA, Complementary - blood
DNA, Complementary - genetics
Doxycycline
Doxycycline - pharmacology
Embryo cells
Embryonic stem cells
Flexibility
Gene expression
Gene Expression Regulation - drug effects
Gene Knockdown Techniques
Gene sequencing
Gene Transfer Techniques
Genes
Genomes
HEK293 Cells
Humans
Lentivirus - genetics
Medical research
Mice
MicroRNAs
Mutagenesis, Insertional - methods
Neurosciences
Pathology
Platforms
Prostate
Recombination
Recombination, Genetic
RNA, Small Interfering - biosynthesis
RNA, Small Interfering - genetics
Rodents
Science
Stem cell transplantation
Stem cells
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0116373