title: Engineered Promoters for Potent Transient Overexpression.
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 gene-therapy-related applications that require potent expression for extended time periods.
authors.affiliation: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, 5290002, Israel.
authors.forename: Dan Y
authors.initials: DY
authors.lastname: Even
authors.affiliation: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, 5290002, Israel.
authors.forename: Adi
authors.initials: A
authors.lastname: Kedmi
authors.affiliation: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, 5290002, Israel.
authors.forename: Shani
authors.initials: S
authors.lastname: Basch-Barzilay
authors.affiliation: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, 5290002, Israel.
authors.forename: Diana
authors.initials: D
authors.lastname: Ideses
authors.affiliation: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, 5290002, Israel.
authors.forename: Ravid
authors.initials: R
authors.lastname: Tikotzki
authors.affiliation: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, 5290002, Israel.
authors.forename: Hila
authors.initials: H
authors.lastname: Shir-Shapira
authors.affiliation: Faculty of Engineering and Bar-Ilan Institute of Nanotechnologies and Advanced Materials, Bar-Ilan University, Ramat-Gan, 5290002, Israel.
authors.forename: Orit
authors.initials: O
authors.lastname: Shefi
authors.affiliation: The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, 5290002, Israel.
authors.forename: Tamar
authors.initials: T
authors.lastname: Juven-Gershon
mesh_terms: D000818:Animals; D003587:Cytomegalovirus / Q000235:genetics; D005434:Flow Cytometry; D015870:Gene Expression; D005814:Genes, Viral; D005818:Genetic Engineering; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D015533:Transcriptional Activation
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins