Interrupting the (GAA•TTC)₁₀₈ repeat with a transposon does not improve transcription <i>in vitro</i>.

<p>The strategy used for <i>in vitro</i> transcription of linear and supercoiled templates is summarized in the schematic at the top. Immediately below that is the image of products of <i>in vitro</i> transcription of linear and of supercoiled plasmids visualized by hybridization with a biotinylated ODN probe followed by chemiluminescence. The templates are as follows: Lane 1∶9 repeats, no transposon. Lane 2∶108 repeats, no transposon. Lane 3∶9 repeats, Tn5 transposon 5′ to the first GAA. Lane 4∶108 repeats, transposon Tn5 86 bp 5′ of the first GAA. Lane 5∶108 repeats, Tn5 transposon at repeat 8. Lane 6∶108 repeats, Tn5 transposon at repeat 57. The intensity of the bands representing <i>in vitro</i> transcription was quantitated with Kodak Molecular Imaging software. Bars represent mean amount of transcription for three independent experiments. A representative blot is shown.</p

The location and orientation of Tn7 insertion within the GAA•TTC repeat is pH dependent.

<p>Depictions of the MluI to XbaI fragment containing the GAA•TTC repeat with vertical lines indicating Tn7 insertion events at pH 7, pH 8 and pH 9 both within and near the repeat. The repeat is divided into thirds, and point deletions and substitutions in the GAA repeat are indicated by open circles and asterisks, respectively. Lines extending above represent insertions events in the (+) orientation, defined as the transposon oriented from left to right. Lines extending below represent insertion events in the (−) orientation with the transposon oriented right to left. Insertions performed at pH 7 and pH 8 show a strong bias for the left to right (+) orientation of the transposon. At pH 7.0 and pH 8.0, Tn7 shows a bias for insertion within the largely uninterrupted regions 1 and 2. In contrast, region 3, an area where the GAA•TTC repeat contains multiple interruptions, is not a favored target for insertion. Location of insertion was determined by DNA sequencing.</p

Preferential insertion into the (GAA•TTC)₁₀₈ repeat by Tn7 is pH dependent.

<p>Bars indicate transposon insertion into the (GAA•TTC)₁₀₈ repeat as a percentage of total insertions in the target plasmid at several pH values. Transposon Tn7 (black bars) shows a pH dependent bias for insertion into the repeat, inserting 48, 39 and 14 percent of the time at pH 7, pH 8 and pH 9, respectively. In contrast, transposon Tn5 is not attracted to the GAA•TTC repeat, inserting 6, 7 and 8 percent of the time at pH 7, pH 8 and pH 9, respectively. Insertion location was determined by a combination of restriction digest mapping and DNA sequencing. The expected frequency based on target size is 9% (gray bar). For pH 7 and pH 9 n = 96, for pH 8 n = 120.</p

Experimental Target.

<p>Plasmid targets for <i>in vitro</i> transposition reactions contained 108 GAA•TTC repeats. Hollow boxes indicate essential regions (ori, <i>bla</i>) of the plasmid excluded from recoverable transposition events. The expected frequency of insertion into the (GAA•TTC)₁₀₈ region based on its contribution to the size of the plasmid (minus the essential regions) is 9%. The repeat is located between restriction sites MluI and XmaI. The repeat is divided into thirds, and point deletions and substitutions in the GAA repeat are indicated by open circles and asterisks, respectively. The sequence of this repeat, divided into thirds, is shown at the bottom of the figure. The complete sequence of the insert is available online (GU722204).</p

Xrn2 and Senataxin knockdowns provide limited release from transcription termination.

<p><i>A</i>, Western blot analysis of Xrn2 knockdown in whole cell extracts. TAN dsred control cell line was grown as indicated in the methods and transfected with a plasmid expressing an shRNA targeting Xrn2. Samples were run on duplicate gels in parallel, blotted and then probed for Xrn2 expression. Anti-Xrn2 antibody (Bethyl Laboratories) produces a doublet, shown here. The lower band disappears with shRNA targeting Xrn2, and the upper band shows reduced density. Anti-human β-actin antibody serves as a control. The analysis was performed three times, with a representative blot shown. <i>B</i>, Xrn2 and senataxin knockdowns each provide little release from transcription termination. Cell lines were grown as detailed in the methods. The y-axis values are hRLUC/FLUC expression ratios normalized to a positive cell lysate run in each plate. Error bars indicate the S.E.M. for a sample number of three. Significant differences (p<0.05) from the corresponding vector (pLKO) treated samples are indicated by asterisks.</p

Runner sequence offers modest release from transcription termination.

<p><i>A</i>, The inclusion of 2.6 kilobases of dsred tetrameric sequence downstream (3′) to the ACT and HBB sequences provides modest release from transcription termination. Transcription was induced with doxycycline and cells harvested after 24 hours. The y-axis values are hRLUC/FLUC expression ratios normalized to a positive cell lysate run in each plate and expressed as a percentage of the dsred control cell line. All of the changes are significant (p<0.05) when compared to the dsred control. The error bars indicate the S.E.M. for a sample number of three. <i>B</i>, Real-time RT-PCR analysis. FLUC and hRLUC mRNA was measured by real-time RT-PCR and expressed as the relative ratio of hRLUC/FLUC mRNA. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006193#s2" target="_blank">Results</a> are shown as a percentage of the dsred control cell line. The ratio decreased as larger tracts of polyadenylation sequence are included in the tandem construct. Error bars indicate the S.E.M. for a sample number of three.</p

The use of tandem reporters to measure transcription elongation.

<p><i>A</i>, A tetracycline-regulated promoter drives transcription through the tandem reporters. Self-cleaving hammerhead ribozymes cut the RNA transcript, separating both FLUC and hRLUC expressing RNA fragments from the test sequence. An internal ribosome entry sequence (IRES) enhances translation of the uncapped hRLUC expression fragment by replacing functions of the 5′ cap and untranslated region (5′ UTR). The relative ratio between the reporters measures the transcriptional impediment presented by an insert. The 5′ reporter (FLUC) is firefly luciferase and the 3′ reporter (hRLUC) is a humanized sea pansy luciferase. If the RNA polymerase terminates transcription in the inserted test sequence, the 3′ reporter will not be transcribed. <i>B</i>, Robust, reproducible induction of FLUC and hRLUC activities in clonal cell lines. Graphs display the mean luciferase activity per cell in Relative Light Units (RLU) for FLUC and hRLUC from clonal cell lines containing a single integrated copy of a control tandem reporter construct. Cells were cultured without (−) or with (+) the addition of doxycycline for 24 hours to induce transcription from the promoter. Extracts representing 15,000 cells were assayed for luciferase activity. The mean induction for FLUC expression was 237±29 fold, and 56±3 for hRLUC. Error bars indicate the S.E.M. for a sample number of three. <i>C</i>, Ribozymes self-cleave in a human cell line. Gel shows yield of RT-PCR products Transcription was induced with doxycycline and RNA was harvested after 24 hours. RT-PCR was conducted using primers designed for FLUC and hRLUC coding regions, and primers designed to span the ribozymes and the 275 bp of linker sequence between the two ribozymes. TAN-NR does not contain self-cleaving ribozymes, and all three sets of primers successfully amplify the target. β-actin primers were included as an amplification control. RT-PCR products were separated on a 1% agarose gel.</p

HBO treatment delays wound closure and blastema formation.

<p>H&E staining of 7, 10, and 14 DPA sections of control (A-C) and HBO-treated (A'-C') digits show cellular changes in the marrow and delayed wound closure and blastema formation in HBO-treated digits. Arrows indicate areas of vasculature on control 7 DPA (A) and HBO treated 7 DPA (A') samples. (B') Epidermis remains open at 10 DPA in samples treated with hyperbaric oxygen. (C') Blastema formation is delayed until 14 DPA in HBO-treated samples when control samples (C) are already forming bone. Higher magnification analysis of the proximal endosteum show activated osteoblasts in HBO-treated samples (A'-C') while control samples return to an inactivated morphology at 10 DPA (B). Scale bars = 100 μm in panels and 5 μm in insets. (N = 8 HBO and N = 7 control with representative figure shown).</p

HBO treatment alters the morphology of regenerated bone.

<p>Polarized light micrographs of control and HBO treated digits at 7, 10, 14, and 21 DPA show differences in collagen fiber composition at distinct time points during regeneration. Collagen fiber alignment and thickness is readily identifiable under polarized light. Thinner fibers (a general indication of either thin collagen I fibrils, <50 nm in width, or collagen III fibers) appear green. Thick, more mature and aligned collagen I fibers appear red or yellow [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140156#pone.0140156.ref022" target="_blank">22</a>]. Photomicrographs are presented in transmitted light (A—F) and polarized light (A'-F'). Digit orientation is portrayed by black cartoon on the upper right corner of each image. Scale bars = 100 μm. N = 3 with representative sample shown.</p

Continuous HBO treatment enhances bone degradation.

<p>(A) Bone regeneration during HBO application shows a similar rate of bone degradation before beginning to grow bone at 12 DPA (N = 4 mice, N = 16 digits). Samples were analyzed for bone growth using μCT. Data are normalized to initial 0 DPA bone volume and analyzed using a SS ANOVA algorithm accounting for variation between the individual mice (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140156#sec006" target="_blank">methods</a>). (B) Representative X-ray images of a regenerating P3 digit at 0, 7, 10, 17, and 28 DPA. (C) 14 DPA time point capture showing the P3 regenerative response in a control digit, (from A). (D) 14 DPA time point capture of a representative digit (from A) treated with continuous HBO twice daily showing increased bone degradation. Full time lapse sequence available in supplemental materials. (E) Effect of daily HBO application on osteoclast numbers at 7, 10, and 14 DPA. Results are expressed as mean ± SEM. ^# P<0.05, comparison of control to HBO. NOc/BPm; number of osteoclasts/bone perimeter. (F) Micro-CT scans of the same control sample seen in C and (G) same HBO sample seen in D are pseudo-colored according to trabecular thickness. (*) Asterisk indicates degradation through the proximal os-hole. Color changes indicate bone thickness in μm.</p