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

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 - 2017/7/1

Y1 - 2017/7/1

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

SN - 0305-1048

VL - 45

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 13

M1 - e123

ER -