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

In this study, the authors combined the use of genome editing tools and last generation inducible systems in order to develop a cell model where the expression of CDX2 transcription factor can be tightly regulated. CDX2 is an intestinal differentiation marker whose expression is often altered in gastric and colon carcinomas. Its control was here shown to be useful to uncover novel putative downstream effector genes (such as GPA33 and LDLR), and therefore, to further clarify the role of CDX2 in intestinal cells. This new strategy can possibly be adapted to any other gene of interest.

As the result of a collaboration between IPATIMUP and the Copenhagen Center for Glycomics (University of Copenhagen), this work has recently been published in Nucleic Acid Research.

Authors and Affiliations:

Pinto R^1,2,3,4, Hansen L⁴, Hintze J⁴, Almeida R^1,2,3,5, Larsen S^6,7, Coskun M^8,9, Davidsen J⁶, Mitchelmore C⁶, David L^1,2,3, Troelsen JT⁶, Bennett EP⁴

¹ i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.

² IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal.

³ Faculty of Medicine of the University of Porto, Porto, Portugal.

⁴ Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and Odontology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.

⁵ Department of Biology, Faculty of Sciences of the University of Porto, Porto, Portugal.

⁶ Department of Science and Environment, Roskilde University, Roskilde, Denmark.

⁷ Department of Clinical Immunology, Naestved Hospital, Naestved, Denmark.

⁸ Department of Gastroenterology, Medical Section, Herlev Hospital, University of Copenhagen, Copenhagen, Denmark.

⁹ The Bioinformatics Centre, Department of Biology & Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark.

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.

Journal: Nucleic Acid Research

Link: https://academic.oup.com/nar/article-lookup/doi/10.1093/nar/gkx371