Engineered Promoters for Potent Transient Overexpression.

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

Flow cytometric analysis of long-term fluorescence intensity and number of fluorescent HeLa S3 and SH-SY5Y cells.

<p>HeLa S3 and SH-SY5Y cells were transiently transfected with pRc/CMV, natural CMV, SCP2 or SCP3 vector expressing <i>EGFP</i>. The cells were collected 4–8 days post-transfection (P.T.) for flow cytometric analysis. (A) Flow cytometric analysis of fluorescence intensity of all HeLa S3 fluorescent cells and SH-SY5Y fluorescent cells. (B) Flow cytometric analysis of fluorescence intensity of high intensity HeLa S3 fluorescent cells and SH-SY5Y fluorescent cells. (C) Flow cytometric analysis of the number of all HeLa S3 fluorescent cells and SH-SY5Y fluorescent cells. (D) Flow cytometric analysis of the number of high intensity HeLa S3 fluorescent cells and SH-SY5Y fluorescent cells. Data shown are representative of 6 independent experiments using HeLa S3 cells and 5 independent experiments using SH-SY5Y cells. Statistical comparisons between the promoters were done using the Kruskal—Wallis test with pairwise comparisons. Significant p-values (p ≤0.05) are indicated in the results section.</p

Live cell <i>EGFP</i> imaging of short-term expression of pRc/CMV-based constructs, in HeLa S3 and SH-SY5Y cells.

<p>HeLa S3 and SH-SY5Y cells were transiently transfected with either the pRc/CMV, natural CMV, SCP2 or SCP3 vector expressing <i>EGFP</i>. The cells were imaged once a day during 1–4 days post-transfection (P.T.). Each circle displays the whole well image constructed by stitching individual microscopic fields. (A) HeLa S3 cells. (B) SH-SY5Y cells. Data shown are representative of 3 independent experiments for each cell type.</p

Schematic representation of the engineered core promoters.

<p>The pRc/CMV vector (Life Technologies) contains the CMV enhancer and TATA box, but lacks any CMV sequences that are downstream of -16 relative to the +1 transcription start site (including the Inr element). Three variants of pRc/CMV were constructed in which the core promoter region (from -36 to +45) was replaced with either the natural CMV core promoter, which contains the CMV TATA and Inr elements, or with SCP2 or SCP3, which contains the CMV TATA and Inr, the <i>Tollo</i> MTE, and the <i>Calm2</i> DPE. Single nucleotide changes in SCP3 (relative to SCP2) are marked by red rectangles. Each of these pRc/CMV-based constructs contains the <i>EGFP</i> reporter gene.</p

Flow cytometric analysis of short-term fluorescence intensity and number of fluorescent HeLa S3 and SH-SY5Y cells.

<p>HeLa S3 and SH-SY5Y cells were transiently transfected with pRc/CMV, natural CMV, SCP2 or SCP3 vector expressing <i>EGFP</i>. The cells were collected 1–4 days post-transfection (P.T.) for flow cytometric analysis. (A) Flow cytometric analysis of fluorescence intensity of all HeLa S3 fluorescent cells and SH-SY5Y fluorescent cells. (B) Flow cytometric analysis of fluorescence intensity of high intensity HeLa S3 fluorescent cells and SH-SY5Y fluorescent cells. (C) Flow cytometric analysis of the number of all HeLa S3 fluorescent cells and SH-SY5Y fluorescent cells. (D) Flow cytometric analysis of the number of high intensity HeLa S3 fluorescent cells and fluorescent SH-SY5Y cells. Data shown are representative of 5 independent experiments using HeLa S3 cells and 6 independent experiments using SH-SY5Y cells. Statistical comparisons between the promoters were done using the Kruskal—Wallis test with pairwise comparisons. Significant p-values (p ≤0.05) are indicated in the results section.</p

A comparison of the four core promoters DNA sequences.

<p>A comparison of the four core promoters DNA sequences.</p

Real-Time quantitative PCR of purified transiently transfected plasmid DNA in HeLa S3 and SH-SY5Y cells.

<p>HeLa S3 and SH-SY5Y cells were transiently transfected with pRc/CMV, natural CMV, SCP2 or SCP3 vector expressing <i>EGFP</i>, and harvested every other day during 8 days post-transfection (P.T.). Plasmid DNA was purified from cells and subjected to qPCR analysis with primers for the GAPDH, <i>EGFP</i> and Neomycin genes. Data shown are the averaged Ct values of 3 independent experiments (each performed in triplicates). (A) 2 days post-transfection. (B) 4 days post-transfection. (C) 6 days post-transfection. (D) 8 days post-transfection. Error bars represent SEM.</p