The Impact of cHS4 Insulators on DNA Transposon Vector Mobilization and Silencing in Retinal Pigment Epithelium Cells

<div><p>DNA transposons have become important vectors for efficient non-viral integration of transgenes into genomic DNA. The <em>Sleeping Beauty</em> (SB), <em>piggyBac</em> (PB), and <em>Tol2</em> transposable elements have distinct biological properties and currently represent the most promising transposon systems for animal transgenesis and gene therapy. A potential obstacle, however, for persistent function of integrating vectors is transcriptional repression of the element and its genetic cargo. In this study we analyze the insulating effect of the 1.2-kb 5′-HS4 chicken β-globin (cHS4) insulator element in the context of SB, PB, and <em>Tol2</em> transposon vectors. By examining transgene expression from genomically inserted transposon vectors encoding a marker gene driven by a silencing-prone promoter, we detect variable levels of transcriptional silencing for the three transposon systems in retinal pigment epithelium cells. Notably, the PB system seems less vulnerable to silencing. Incorporation of cHS4 insulator sequences into the transposon vectors results in 2.2-fold and 1.5-fold increased transgene expression levels for insulated SB and PB vectors, respectively, but an improved persistency of expression was not obtained for insulated transgenes. Colony formation assays and quantitative excision assays unveil enhanced SB transposition efficiencies by the inclusion of the cHS4 element, resulting in a significant increase in the stable transfection rate for insulated SB transposon vectors in human cell lines. Our findings reveal a positive impact of cHS4 insulator inclusion for SB and PB vectors in terms of increased transgene expression levels and improved SB stable transfection rates, but also the lack of a long-term protective effect of the cHS4 insulator against progressive transgene silencing in retinal pigment epithelium cells.</p> </div

Enhanced Tailored MicroRNA Sponge Activity of RNA Pol II-Transcribed TuD Hairpins Relative to Ectopically Expressed ciRS7-Derived circRNAs

As key regulators of gene expression, microRNAs (miRNAs) have emerged as targets in basic experimentation and therapy. Administration of DNA-encoded RNA molecules, targeting miRNAs through base pairing, is one viable strategy for inhibiting specific miRNAs. A naturally occurring circular RNA (circRNA), ciRS-7, serving as a miRNA-7 (miR-7) sponge was recently identified. This has sparked tremendous interest in adapting circRNAs for suppressing miRNA function. In parallel, we and others have demonstrated efficacy of expressed anti-miRNA Tough Decoy (TuD) hairpins. To compare properties of such inhibitors, we express ciRS-7 and TuD-containing miRNA suppressor transcripts from identical vector formats adapted from RNA polymerase II-directed expression plasmids previously used for production of ciRS-7. In general, markedly higher levels of miR-7 suppression with TuD transcripts relative to ciRS-7 are observed, leading to superior miRNA sponge effects using expressed TuD hairpins. Notably however, we find that individual ciRS-7 transcripts are more potent inhibitors of miR-7 activity than individual TuD7-containing transcripts, although each miR-7 seed match target site in ciRS-7 is, on average, less potent than the perfectly matched target sites in the TuD motif. All together, our studies call for improved means of designing and producing circRNAs for customized miRNA targeting to match TuD hairpins for tailored miRNA suppression. Keywords: miRNA suppression, TuD, miRNA sponge, circular RNA, ciRS-

Silencing of SB, PB, and <i>Tol2</i> transposon-based vectors in ARPE-19 cells.

<p>(<b>A</b>) Percentage of retained median fluorescence intensity (MFI) for stably transfected ARPE-19 clones. ARPE-19 cells were transfected with pSBT/RGIP, pPBT/RGIP, or pTol2T/RGIP together with a transposase expressing plasmid, and selected for puromycin resistance. Individual clones were expanded and then passaged for 8 weeks in the absence of selection. Their eGFP expression level was determined at day 0 and day 56 of passage, and their percentage of retained MFI was calculated. (<b>B</b>) Percentage of retained median fluorescence intensity (MFI) for stably transfected ARPE-19 clones containing 1–3 transposon insertions. (<b>C</b>) Percentage of retained median fluorescence intensity (MFI) for stably transfected ARPE-19 clones containing 9 or more transposon insertions. (<b>D</b>) Reactivation of eGFP expression by TSA treatment. A subset of silenced ARPE-19 cell clones was grown in the presence of the deacetylase inhibitor Trichostatin A (TSA). The clones were treated 24 hours before analysis of eGFP expression by flow cytometry.</p

Insulation of SB, PB, and Tol2 transposon vectors in ARPE-19 cells.

<p>(<b>A</b>) Percentage of retained median fluorescence intensity (MFI) for stably transfected ARPE-19 clones carrying insulated transposon vectors. Measurements were obtained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048421#pone-0048421-g002" target="_blank">Figure 2a</a>. (<b>B</b>) Comparison of mean MFI levels for insulated and uninsulated clones. Stably transfected ARPE-19 cell clones were grown for 8 weeks in the absence of selection, and eGFP expression levels were measured by flow cytometry at day 0 and day 56 of passage. (<b>C</b>) Comparison of mean MFI levels for insulated and uninsulated clones carrying 1-3 transposon insertions. (<b>D</b>) Comparison of mean MFI levels for insulated and uninsulated clones carrying 9 or more transposon insertions.</p

Transposition of SB, PB, and <i>Tol2</i> transposon vectors in ARPE-19 cells.

<p>(<b>A</b>) Schematic representation of pSBT/RGIP, pPBT/RGIP, and pTol2T/RGIP vectors. IR, inverted repeat; RSV, Rous sarcoma virus promoter; eGFP, enhanced green fluorescent protein; IRES, internal ribosome entry site; puro, puromycin resistance gene; pA, polyadenylation site. (<b>B</b>) Stable transfection rates of SB, PB, and <i>Tol2</i> transposon vectors in ARPE-19 cells. 0.125 pmol of pSBT/RGIP, pPBT/RGIP, and pTol2T/RGIP plasmid were cotransfected together with 0.02 pmol pcDNA3.1D/V5.TOPO plasmid (empty vector) or 0.02 pmol helper plasmid expressing either <i>SB100X</i> transposase, <i>iPB</i> transposase, or <i>Tol2</i> transposase. The pcDNA3.1D/V5.TOPO plasmid was also included as non-specific DNA to ensure that the total amount of DNA was 1 µg in each transfection. After 8 days of selection, puromycin resistant colonies were stained and counted. Mean ± SEM values are shown (N = 3). P values listed above the brackets were obtained by student's t-tests. (<b>C</b>) Transposon copy number of stably transfected ARPE-19 clones. Genomic DNA from ARPE-19 cell clones carrying SBT/RGIP, PBT/RGIP, or Tol2T/RGIP transposons was purified and examined by Southern blot analysis to determine the transposon copy number. A representative Southern blot is shown.</p