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

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

A comparison of the four core promoters DNA sequences.

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