Association of human MARs with a specific chromatin pattern.

<p>A) 1683 predicted human MAR genomic locations were aligned using the central positions of their AT rich cores. ChiP-Seq profiles were calculated over the MAR collection for the histone modifications H3K4me3, H3K27me3, H3K36me3 and for RNA Polymerase II. (B) 25000 RefSeq promoters were aligned at their respective TSS positions and oriented according to the direction of transcription. ChiP-Seq profiles were calculated over the promoter collection for indicated histone modification, and for the RNA Pol II. Tag counts were normalized globally and they are expressed as a fold change over the non-precipitated input DNA profile.</p

Identification of the portions of MAR 1–68 that contribute to the anti-silencing and transcriptional effects.

<p>The AT core extended region of the MAR 1–68, as well as a series of sub-fragments of the 5′ and 3′ flanking regions, were cloned upstream of the EGFP reporter gene in both orientation and analyzed for their effects on GFP expression levels. Constructs containing the full-length MAR 1–68 or a control spacer DNA cloned upstream of the EGFP reporter gene were also transfected as controls. GFP fluorescence was measured by flow-cytometry on polyclonal cell pools obtained after 2 weeks of antibiotic selection following transfection, and the proportion of silent and of high expressor cells were scored as illustrated in Fig. 1B. Results illustrate the mean and standard deviation of 3 independent experiments. Significant differences relative to the corresponding control construct containing spacer DNA of the same size, as illustrated in Suppl. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079262#pone.0079262.s003" target="_blank">Fig. S3</a>, are indicated by stars above each bar, whereas line-associated stars indicate significant differences with constructs containing the full length MAR 1–68 or its extended core (Student test, P<0.05).</p

Relative contribution of MAR AT-rich cores and flanking sequences to the anti-silencing and transcriptional effects.

<p>The contribution of the AT rich DNA sequences of MAR 1–68 and X-29 alone (A), or combinations of the MAR 1–68 core with portions of its flanking sequences (B), were assessed for their anti-silencing and transcriptional augmentation activities as described in the legend to Fig. 2. An oligomeric form of the X-29 AT-rich region, consisting of three tandem repeats, was also analyzed. Results represent the mean±SD of 3 independent experiments and the statistical analysis are as for Fig. 2.</p

Schematic representation of MAR 1–68 subdomains and illustration of its anti-silencing and transcriptional effects.

<p>(A) Schematic diagram representing the full-length human MAR 1–68 and its series of sub-fragments, cloned upstream of a minimal SV40 promoter and EGFP reporter gene. The 3.6 kb MAR 1–68 was subdivided into three regions: The MAR 1–68 “extended AT core” region encompassing the AT dinucleotide-rich sequence (yellow box, labelled A), its 5′ (blue, labelled B) and 3′ (green, labelled C) adjacent regions. Putative transcription factor binding sites for the SATB1, NMP4, CEBP, Fast and Hox transcription factors are illustrated by ellipses. The 5′ and 3′ flanking regions were further divided in portions comprising nt 1–910 (labelled D), nt 864–1652 (E), nt 2444–3000 (F) and nt 3020–3628 (G). (B) A typical flow cytometry profile of CHO DG44 cells stably co-transfected with the GFP expression vector containing full-length human MAR 1–68 (black line) or control spacer DNA (no MAR, red line) and with a neomycin resistance plasmid. 10⁵ cells were subjected to flow cytometry analysis for GFP expression after 2 weeks of nemomycin selection. Cells displaying background fluorescence (silent cells) or high GFP expression levels are as indicated.</p

Transposition assay in mammalian cells.

<p>(A) Donor plasmid harboring the transposable element. This vector is composed of the neomycin phosphotransferase gene (Neo^R) flanked by the <i>PB</i> ITRs. (B) Transposition efficiency by type and quantity of nucleic acid. Cells were transfected with indicated amounts of <i>PB</i> mRNA or pDNA alongside with 187.5 ng of donor plasmid. GFP mRNA or pDNA (500 ng) served as negative controls of transposition (Mock: recombination events). To consider only transposition events, the number of colonies observed in the presence of the transposase source was subtracted by the number of colonies obtained in the respective negative control performed without transposase for each quantity. Values represent the average of 3 experiments done in triplicate. * Represents statistically significant difference between mRNA and pDNA (p<0.05).</p

<i>piggyBac</i> transposase bioavailability.

<p>(<b>A</b>) <b>Half-life of the <i>PB</i> mRNA</b>. Cells were transfected with 187.5 ng of <i>PB</i> mRNA. Total RNA was extracted at indicated times, reverse transcribed and subjected to qPCR. 18S RNA served as an internal standard to normalize the data. (<b>B</b>) <b>Persistence of <i>PB</i> pDNA after transfection</b>. Cells were transfected with 187.5 ng of <i>PB</i> pDNA and plasmid rescue was performed at 0 to 20 days. Ampicillin-resistant colonies were selected to evaluate the persistence of the plasmid. (<b>C</b>) <b>Half-life of the <i>PB</i> transposase (V5PB Tp)</b>. Cells were transfected with 187.5 ng of <i>PB</i> mRNA, incubated 18 h to reach the peak of transposase expression and treated with cycloheximide (t0=100). Total protein extraction was done at the indicated times from t0. The transposase half-life was determined by Western-Blot and quantification was normalized to the endogenous actin protein.</p

Expression (A) and kinetic (B) of <i>PB</i> transposase by type and quantity of nucleic acid.

<p>HeLa cells were transfected with indicated amounts (A) or 187.5 ng (B) of <i>PB</i> mRNA or pDNA and total protein extraction was performed 24 h (A) or at indicated times (B) post-transfection. Transposase (V5PB Tp) expression was determined by Western-Blot and protein quantification was normalized to the endogenous Menin protein. Values represent the average of 3 experiments done in triplicate. Mock: untransfected cells. * Indicates statistically significant differences between mRNA and pDNA (p<0.05).</p

Detection of H2AX phosphorylation following dose-dependent <i>PB</i> mRNA or pDNA transfection.

<p>Cells were transfected with indicated amounts of <i>PB</i> mRNA or pDNA and total protein extraction was performed 24 h post-transfection. γ-H2AX expression was determined by Western-Blot and protein quantification was normalized to the endogenous Menin protein. Mock: untransfected cells. T-_GFP: cells transfected with 500 ng of GFP mRNA or pDNA. T+: untransfected cells treated with 2 µg/mL doxorubicin (positive control). * Indicates statistically significant differences between treated and untreated cells (p<0.05). Values represent the average of 3 experiments done in triplicate. The signal between the dotted line (mock control) and solid line (T-_GFP control) is considered to be due to the “transfection effect”. Above the solid line, the signal is due to the “transposase effect”.</p

Effect of MAR on transposon copy number.

<p>The copy number of integrated GFP was determined using qPCR as described in supplementary. Fig S4, using unselected puromycin-resistant cells generated as described in the legends of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062784#pone-0062784-g002" target="_blank">Figure 2</a>. The mean GFP fluorescence was divided by the number of integrated GFP transgene copies, to estimate the average expression per integrated GFP copy in unselected (<b>A</b>) and puromycin-resistant (<b>B</b>) CHO cells. Values represent the means ± SEM (n = 3). *P<0.05.</p

Recombinant protein expression from electroporated CHO-M cell suspensions.

<p>(<b>A</b>) CHO-M cell were electroporated once or twice with the MAR X-29-bearing GFP-expression transposon vector in the presence (+PB) or not (-PB) of the piggyBac transposase, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062784#pone.0062784.s005" target="_blank">Figure S5</a>, and the percentage of stable GFP-expressing cells was assayed after 3 weeks of culture performed in the absence of selection. (<b>B</b>) Mean of the GFP fluorescence of the GFP-positive cells. (<b>C</b>) cDNAs encoding immunoglobulin light and heavy chains of the Bevacizumab (Beva), Adalimumab (Adal) and Rituximab (Ritu) antibody were introduced in MAR X29-containing transposon plasmids instead of GFP. The light and heavy chain transposon constructs were electroporated three times at 12 days intervals with the piggyBac transposase expression vector in CHO-M cells, and the levels of immunoglobulin secreted in the culture supernatants of polyclonal cell pools grown without selection was assayed (open bars). Alternatively, the unselected polyclonal cell populations were sorted by panning cells displaying immunoglobulins at their surface using magnetic micro-beads, and the levels of secreted immunoglobulins were assayed as for the unsorted populations (closed bars). (<b>D</b>) Alternatively, immunoglobulin-expressing colonies were sorted from transfected cell populations using a colony-picking device, and two clones expressing each of the three immunoglobulins were grown in fed-batch cultures in spin-tube bioreactors. The levels of secreted immunoglobulins were determined as for panel (C).</p