Precise integration of inducible transcriptional elements (PrIITE) enables absolute control of gene expression

Rita Pinto, Lars Hansen, John Hintze, Raquel Almeida, Sylvester Larsen, Mehmet Coskun, Johanne Davidsen, Cathy Mitchelmore, Leonor David, Jesper Troelsen, Eric Paul Bennett

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

Tetracycline-based inducible systems provide powerful methods for functional studies where gene expression can be controlled. However, the lack of tight control of the inducible system, leading to leakiness and adverse effects caused by undesirable tetracycline dosage requirements, has proven to be a limitation. Here, we report that the combined use of genome editing tools and last generation Tet-On systems can resolve these issues. Our principle is based on precise integration of inducible transcriptional elements (coined PrIITE) targeted to: (i) exons of an endogenous gene of interest (GOI) and (ii) a safe harbor locus. Using PrIITE cells harboring a GFP reporter or CDX2 transcription factor, we demonstrate discrete inducibility of gene expression with complete abrogation of leakiness. CDX2 PrIITE cells generated by this approach uncovered novel CDX2 downstream effector genes. Our results provide a strategy for characterization of dose-dependent effector functions of essential genes that require absence of endogenous gene expression.
OriginalsprogEngelsk
TidsskriftNucleic Acids Research
Vol/bind45
Udgave nummer13
Sider (fra-til)e123
Antal sider15
ISSN0305-1048
DOI
StatusUdgivet - 4 maj 2017

Citer dette

Pinto, Rita ; Hansen, Lars ; Hintze, John ; Almeida, Raquel ; Larsen, Sylvester ; Coskun, Mehmet ; Davidsen, Johanne ; Mitchelmore, Cathy ; David, Leonor ; Troelsen, Jesper ; Bennett, Eric Paul. / Precise integration of inducible transcriptional elements (PrIITE) enables absolute control of gene expression. I: Nucleic Acids Research. 2017 ; Bind 45, Nr. 13. s. e123.
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title = "Precise integration of inducible transcriptional elements (PrIITE) enables absolute control of gene expression",
abstract = "Tetracycline-based inducible systems provide powerful methods for functional studies where gene expression can be controlled. However, the lack of tight control of the inducible system, leading to leakiness and adverse effects caused by undesirable tetracycline dosage requirements, has proven to be a limitation. Here, we report that the combined use of genome editing tools and last generation Tet-On systems can resolve these issues. Our principle is based on precise integration of inducible transcriptional elements (coined PrIITE) targeted to: (i) exons of an endogenous gene of interest (GOI) and (ii) a safe harbor locus. Using PrIITE cells harboring a GFP reporter or CDX2 transcription factor, we demonstrate discrete inducibility of gene expression with complete abrogation of leakiness. CDX2 PrIITE cells generated by this approach uncovered novel CDX2 downstream effector genes. Our results provide a strategy for characterization of dose-dependent effector functions of essential genes that require absence of endogenous gene expression.",
author = "Rita Pinto and Lars Hansen and John Hintze and Raquel Almeida and Sylvester Larsen and Mehmet Coskun and Johanne Davidsen and Cathy Mitchelmore and Leonor David and Jesper Troelsen and Bennett, {Eric Paul}",
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Precise integration of inducible transcriptional elements (PrIITE) enables absolute control of gene expression. / Pinto, Rita; Hansen, Lars; Hintze, John ; Almeida, Raquel; Larsen, Sylvester; Coskun, Mehmet; Davidsen, Johanne; Mitchelmore, Cathy; David, Leonor; Troelsen, Jesper; Bennett, Eric Paul.

I: Nucleic Acids Research, Bind 45, Nr. 13, 04.05.2017, s. e123.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Precise integration of inducible transcriptional elements (PrIITE) enables absolute control of gene expression

AU - Pinto, Rita

AU - Hansen, Lars

AU - Hintze, John

AU - Almeida, Raquel

AU - Larsen, Sylvester

AU - Coskun, Mehmet

AU - Davidsen, Johanne

AU - Mitchelmore, Cathy

AU - David, Leonor

AU - Troelsen, Jesper

AU - Bennett, Eric Paul

PY - 2017/5/4

Y1 - 2017/5/4

N2 - Tetracycline-based inducible systems provide powerful methods for functional studies where gene expression can be controlled. However, the lack of tight control of the inducible system, leading to leakiness and adverse effects caused by undesirable tetracycline dosage requirements, has proven to be a limitation. Here, we report that the combined use of genome editing tools and last generation Tet-On systems can resolve these issues. Our principle is based on precise integration of inducible transcriptional elements (coined PrIITE) targeted to: (i) exons of an endogenous gene of interest (GOI) and (ii) a safe harbor locus. Using PrIITE cells harboring a GFP reporter or CDX2 transcription factor, we demonstrate discrete inducibility of gene expression with complete abrogation of leakiness. CDX2 PrIITE cells generated by this approach uncovered novel CDX2 downstream effector genes. Our results provide a strategy for characterization of dose-dependent effector functions of essential genes that require absence of endogenous gene expression.

AB - Tetracycline-based inducible systems provide powerful methods for functional studies where gene expression can be controlled. However, the lack of tight control of the inducible system, leading to leakiness and adverse effects caused by undesirable tetracycline dosage requirements, has proven to be a limitation. Here, we report that the combined use of genome editing tools and last generation Tet-On systems can resolve these issues. Our principle is based on precise integration of inducible transcriptional elements (coined PrIITE) targeted to: (i) exons of an endogenous gene of interest (GOI) and (ii) a safe harbor locus. Using PrIITE cells harboring a GFP reporter or CDX2 transcription factor, we demonstrate discrete inducibility of gene expression with complete abrogation of leakiness. CDX2 PrIITE cells generated by this approach uncovered novel CDX2 downstream effector genes. Our results provide a strategy for characterization of dose-dependent effector functions of essential genes that require absence of endogenous gene expression.

U2 - 10.1093/nar/gkx371

DO - 10.1093/nar/gkx371

M3 - Journal article

VL - 45

SP - e123

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 13

ER -