Hypothetical model to explain the inversion of 4.8

<p>Step 1: Integration of a NILV mediated by phiC31-int into a p<i>attP</i> site. Step2: Recombination mediated by phiC31-int between the p<i>attL</i> generated during step 1 and another p<i>att</i> site located at 4 kb.</p

Detection of recombination mediated by phiC31-int between an <i>attB</i> site contained into a NILV and a genomic <i>attP</i> site.

<p>A) Scheme of the DsRed2 PCR before and after the enzymatic restriction treatment. B) PCR DsRed2 results without restriction enzyme treatment. Lanes 1 to 3: cotransduction with CMV-Neo and CMV-PhiC31 increasing vector input of 50–150–300 ng of p24. Lanes 4 to 6: cotransduction with <i>attB</i>-CMV-Neo and CMV-PhiC31 increasing vector input of 50–150–300 ng of p24. Lane 7: <i>attB</i>-CMV-Neo. Lane 8: positive control generated by triple-transfection (CMV-phiC31-int, <i>attB</i>-CMV-Neo and CMV-<i>attP</i>-DsRed2). Lane 9: negative control without vector. Lane 10: negative control of PCR. C) PCR DsRed2 results after restriction enzyme treatment. Lanes are similar to figure B. D) Nested PCR from the product isolated from lane 6 to confirm the specificity of PCR DsRed2 amplification.</p

Analysis strategies to detect the specific integrations mediated by phiC31-int.

<p>A) Illustration of the three mechanisms of the phiC31-int mediated integration of a NILV containing an <i>attB</i> sequence. According to the type of integration, the PCR results in three different profiles: - PCRs LTR+/<i>attB</i>− : integration type (1), specific integration. - PCRs LTR−/<i>attB</i>+: integration type (2), residual integration. - PCRs LTR+/<i>attB</i>+: integration type (3), illegitimate integration. P1/P1′ are the primers used for <i>attB</i> PCR and P2/P2′ are the primers used for LTR PCR. B) Schematic representations of the inverse PCR and the adapted inverse PCR strategies used to characterize phiC31-int integration sites.</p

Mapping and description of pa<i>ttP</i> sites isolated by iPCR on human and murine cell lines.

<p>Mapping and description of pa<i>ttP</i> sites isolated by iPCR on human and murine cell lines.</p

Scheme of phiC31-int mediated recombination in bacterial host.

<p>PhiC31 integrase performs precise recombination between an <i>attB</i> site located in the <i>Streptomyces</i> genome and an <i>attP</i> site located on the phiC31 phage genome. The outcome is integration of the phage into the host genome.</p

Analysis of cell lines which constitutively expressed phiC31-int.

<p>A) PhiC31 RT-PCR on three different cell lines. HFi and Hi16 are derived from Hela cell line and TC1 from NIH-3T3 cell line. Control condition lane lacks RNA. B) PCR which detects LTR junctions or intact <i>attB</i> sites after transduction with a NILV <i>attB</i>-CMV-Neo.</p

Repartition in 3 groups of human and murine clones according to the <i>attB</i> and LTR PCR results.

<p>Repartition in 3 groups of human and murine clones according to the <i>attB</i> and LTR PCR results.</p

DNA sequence of <i>att</i> and p<i>attP</i> sites.

<p>A) Wild type <i>attP</i> and <i>attB</i> sites. After recombination two hybrids sites are formed: <i>attL</i> and <i>attR</i>. B) Recombination between <i>attB</i> site and the human locus Xq22.1 This recombination generates a p<i>attR</i> which has been isolated by inverse PCR. Xq22.1 had been described previously as a human p<i>attP</i> by MP Calos et al., who isolated the same p<i>attR</i>.</p

Effect of NLS sequence on phiC31-int activity in NILV context.

<p>A) Cotransduction of NILVs CMV-PhiC31 and CMV-Neo or <i>attB</i>-CMV-Neo. Four p24 doses of PhiC31 vector were used (D1: 3 ng, D2: 5 ng, D3: 10 ng, D4: 33 ng). B) Cotransduction of NILVs CMV-PhiC31-NLS and CMV-Neo or <i>attB</i>-CMV-Neo. Four p24 doses of PhiC31 vector were used (D1: 3 ng, D2: 5 ng, D3: 10 ng, D4: 33 ng). No significant differences are observed between sample with or without a<i>ttB</i> sequence in the vector pTRIP-CMV-Neo. Satistics: two ways ANOVA with Bonferroni posttest (Prism 5).</p

Consequences of AOX expression on mitochondrial properties in the MitAOX mouse.

<p>A, ADP/O values in cortex mitochondria oxidizing succinate. Predicted value (grey) estimated for a 30% cyanide-resistant succinate oxidation (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003182#pgen-1003182-g004" target="_blank">Figure 4A</a>) and full operation of the AOX. B, Oxygen uptake by 100 µg cortex mitochondria oxidizing 10 mM succinate in the presence of 500 µM ADP, in the absence (a) or presence (b) of KCN. Numbers along the traces are nmol/min/mg protein Inset: cyanide-resistance plotted <i>versus</i> oxygen concentration. C, Simultaneous measurement of oxygen consumption (a, c) and reduction of the Amplex Red dye (b, d) by 300 µg cortex mitochondria oxidizing succinate as described under material and methods. Numbers along traces refer to oxygen uptake (a, c) or hydrogen peroxide (b, d) production. D, bar graph indicating the mean value (four independent experiments) ± SD of H₂O₂ production by WT and MitAOX brain mitochondria oxidizing succinate in the presence of antimycin as in C. E, Anesthetized MitAOX and WT mice were exposed to cyanide and survival time noted. Expressed as a percent of WT mice survival time, data for each MitAOX mouse tested were plotted as a function of AOX/ATPase protein levels in brain and lungs, quantitated from Western blots.</p