Precise integration of inducible transcriptional elements (PrIITE) enables absolute control of gene expression
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Precise integration of inducible transcriptional elements (PrIITE) enables absolute control of gene expression. / Pinto, Rita; Hansen, Lars; Hintze, John Birger Hjalmar; Almeida, Raquel; Larsen, Sylvester; Coskun, Mehmet; Davidsen, Johanne; Mitchelmore, Cathy; David, Leonor; Troelsen, Jesper Thorvald; Bennett, Eric Paul.
In: Nucleic Acids Research, Vol. 45, No. 13, e123, 2017.Research output: Contribution to journal › Journal article › Research › peer-review
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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 Birger Hjalmar
AU - Almeida, Raquel
AU - Larsen, Sylvester
AU - Coskun, Mehmet
AU - Davidsen, Johanne
AU - Mitchelmore, Cathy
AU - David, Leonor
AU - Troelsen, Jesper Thorvald
AU - Bennett, Eric Paul
N1 - © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
PY - 2017
Y1 - 2017
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
C2 - 28472465
VL - 45
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 13
M1 - e123
ER -
ID: 179041306