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Inducible and reversible lentiviral and Recombination Mediated Cassette Exchange (RMCE) systems for controlling gene expression
- Source :
- PLoS ONE, Vol 10, Iss 3, p e0116373 (2015), PLoS ONE
- Publication Year :
- 2015
- Publisher :
- Public Library of Science (PLoS), 2015.
-
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.
- Subjects :
- DNA, Complementary
lcsh:Medicine
Biology
Collagen Type I
Cell Line
Small hairpin RNA
Mice
Cell Line, Tumor
Gene Knockdown Techniques
polycyclic compounds
Animals
Humans
Site-specific recombinase technology
RNA, Small Interfering
lcsh:Science
Recombination, Genetic
Regulation of gene expression
Gene knockdown
Multidisciplinary
Recombinase-mediated cassette exchange
Lentivirus
HEK 293 cells
lcsh:R
Gene Transfer Techniques
Molecular biology
Cell biology
Collagen Type I, alpha 1 Chain
Mutagenesis, Insertional
HEK293 Cells
Gene Expression Regulation
Doxycycline
Ectopic expression
lcsh:Q
Research Article
Subjects
Details
- Language :
- English
- ISSN :
- 19326203
- Volume :
- 10
- Issue :
- 3
- Database :
- OpenAIRE
- Journal :
- PLoS ONE
- Accession number :
- edsair.doi.dedup.....8db9475e4e06ec920a28263719bfb9c1