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

PloS One
David C BerstenMurray 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-ind...Continue Reading

References

Jun 25, 2004·Nucleic Acids Research·Ye DingCharles E Lawrence
Sep 6, 2005·Proceedings of the National Academy of Sciences of the United States of America·Frank StegmeierStephen J Elledge
Jan 10, 2006·Genesis : the Journal of Genetics and Development·Caroline BeardRudolf Jaenisch
Jul 29, 2006·Bioinformatics·Chuong B DoSerafim Batzoglou
Sep 2, 2006·Proceedings of the National Academy of Sciences of the United States of America·Kum-Joo ShinIain D C Fraser
Jan 18, 2007·PloS One·Christopher S Raymond, Philippe Soriano
Apr 5, 2007·Nature Protocols·Gustavo TiscorniaInder M Verma
Sep 15, 2007·The Journal of Neuroscience : the Official Journal of the Society for Neuroscience·Shiaoching GongCharles R Gerfen
Sep 26, 2008·Nature·Yingxi LinMichael E Greenberg
Nov 20, 2008·Proceedings of the National Academy of Sciences of the United States of America·Marco J HeroldHolger M Reichardt
Apr 14, 2009·Nature Methods·Daniel G GibsonHamilton O Smith
Dec 17, 2009·Nature Methods·Bryce W CareyRudolf Jaenisch
Jul 10, 2010·Human Gene Therapy·Cheryl Y BrownSimon C Barry
Nov 26, 2010·BMC Biotechnology·Rainer LoewManfred Gossen
Feb 11, 2011·Proceedings of the National Academy of Sciences of the United States of America·Kristen L MeerbreyStephen J Elledge
Apr 6, 2011·Proceedings of the National Academy of Sciences of the United States of America·Bosiljka TasicLiqun Luo
Jun 28, 2011·Nature Methods·Xiaoping YangDavid E Root
Nov 15, 2011·Nucleic Acids Research·Janan T EppigUNKNOWN Mouse Genome Database Group
Jan 6, 2012·Proceedings of the National Academy of Sciences of the United States of America·Xu TanStephen J Elledge
Feb 4, 2012·Nature Protocols·Lukas E DowScott W Lowe
Sep 7, 2012·Frontiers in Genetics·Olga V MatveevaSvetlana A Shabalina
Dec 18, 2012·Nucleic Acids Research·Simone SciabolaRobert V Stanton
Feb 22, 2013·Proceedings of the National Academy of Sciences of the United States of America·Benedikt WefersRalf Kühn
Jun 20, 2013·The Journal of Clinical Investigation·Shwetha RamachandrappaI Sadaf Farooqi
Nov 23, 2013·Nature Reviews. Cancer·David C BerstenMurray L Whitelaw
Dec 3, 2013·Biochimica Et Biophysica Acta·David C BerstenMurray L Whitelaw
Dec 18, 2013·Cell Reports·Christof FellmannJohannes Zuber

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Citations

Nov 18, 2016·Scientific Reports·Karim BenabdellahFrancisco Martin

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Methods Mentioned

BETA
gene knockouts
PCR
transfection
Assay
fluorescence microscopy
genotyping
FACS
transgenic
gene knockdown

Software Mentioned

FlowJo
mir
sfold
R
contrafold
sirna
GraphPad Prism
ImageJ
scan
GraphPad

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