Engineered promoters for potent transient overexpression

Dan Y. Even, Adi Kedmi, Shani Basch-Barzilay, Diana Ideses, Ravid Tikotzki, Hila Shir-Shapira, Orit Shefi, Tamar Juven-Gershon

Research output: Contribution to journalArticlepeer-review

25 Scopus citations

Abstract

The core promoter, which is generally defined as the region to which RNA Polymerase II is recruited to initiate transcription, plays a pivotal role in the regulation of gene expression. The core promoter consists of different combinations of several short DNA sequences, termed core promoter elements or motifs, which confer specific functional properties to each promoter. Earlier studies that examined the ability to modulate gene expression levels via the core promoter, led to the design of strong synthetic core promoters, which combine different core elements into a single core promoter. Here, we designed a new core promoter, termed super core promoter 3(SCP3), which combines four core promoter elements (the TATA box, Inr, MTE and DPE) into a single promoter that drives prolonged and potent gene expression. We analyzed the effect of core promoter architecture on the temporal dynamics of reporter gene expression by engineering EGFP expression vectors that are driven by distinct core promoters. We used live cell imaging and flow cytometric analyses in different human cell lines to demonstrate that SCPs, particularly the novel SCP3, drive unusually strong long-term EGFP expression. Importantly, this is the first demonstration of long-term expression in transiently transfected mammalian cells, indicating that engineered core promoters can provide a novel non-viral strategy for biotechnological as well as genetherapy-related applications that require potent expression for extended time periods.

Original languageEnglish
Article numbere0148918
JournalPLoS ONE
Volume11
Issue number2
DOIs
StatePublished - Feb 2016

Bibliographical note

Publisher Copyright:
© 2016 Even et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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