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

Research output: Contribution to journalJournal articleResearchpeer-review

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

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.

Original languageEnglish
Article numbere123
JournalNucleic Acids Research
Volume45
Issue number13
Number of pages15
ISSN0305-1048
DOIs
Publication statusPublished - 2017

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