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.

Measurement of Rapid Protein Diffusion in the Cytoplasm by Photo-Converted Intensity Profile Expansion

“…Constructs containing Hsp70-SBD and Hsp70-ATPase sequences were kindly provided by Richard Morimoto’s lab (Northwestern University). CL1 sequence was fused to the C terminus of DDR vector pDendra2-C, and the CMV promoter of pDendra2-C was exchanged for the stronger SCP3 promoter (Even et al., 2016), which enhanced expression by 2- to 4-fold compared to CMV (Figure S2). In addition, using pDendra2-C, three constructs with a variable number of DDR repeats (1, 3, and 6, denoted 1xDDR, 3xDDR, and 6xDDR) were cloned.…”

Enzymatic Protein Biopolymers as a Tool to Synthetize Eukaryotic Messenger Ribonucleic Acid (mRNA) with Uses in Vaccination, Immunotherapy and Nanotechnology

“…We have also been attempting to assemble PICs from fission yeast by using whole cell extracts, which contain a full set of TFIIs, RNAPII and regulatory factors (Maldonado, E., unpublished results). For those experiments we used a DNA template containing a super core promoter (SCP) based on the work of Gershon and collaborators [36] and we added a GAL4 binding site upstream of the TATA box to allow strong in vitro transcription activation by the powerful GAL4-VP16 chimeric transcription factor. Those PICs have been assembled and further purified by the use of glycerol gradient sedimentation (Figure 7).…”

Whole synthetic pathway engineering of recombinant protein production

“…We hypothesized that the output from 100RPU.1 could be enhanced by either increasing the number of TFBSs in the proximal region (limited to 14 in the original design), or replacing the nonengineered minimal core region (taken from the hCMV-IE1 promoter). We therefore (a) constructed a hybrid promoter (100RPU_PH) containing the proximal regions from both 100RPU.1 and a second highly active synthetic part, 100RPU.2 (Brown et al, 2017), and (b) created elements (100RPU.1_SC2, 100RPU.1_SC3, and 100RPU.1_SC4) where the hCMV-IE1 core promoter was substituted by varying "super cores" that are specifically designed to maximize transcription initiation rates (Even et al, 2016;Juven-Gershon et al, 2006; see Supporting Information Figure S1).…”