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

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

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

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

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

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

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

Case reports: pig 917.

<p>Grouped data for the transgenic pig 917 (3 integrated copies). (A) Photopic ERG (single flash 3 and 10 cds/m² and 30 Hz flicker) at 11 and 52 weeks. (B) Behavioural observation for pig 917 (red opened circle) compared to non-transgenic control animals (black lozenges, mean and SEM depicted). (C) Histological quantification compared to the mean ± SEM of the non-transgenic controls. (D) RT-PCR analysis of transgene, endogenous GUCY2D and GAPDH gene expression in the retina. (E–P) Immunolabeling for M-opsin (E,I), PNA (F,J) and merged picture (G,K), GFAP (H,I) and S-opsin (I,J,O,P) in the central region of the retina of pig 917 (E–J) and a non-transgenic control (K–P). Arrows in E, G, K and M show examples of M-opsin positive outersegment, arrowhead in G shows displaced nuclei, arrowhead in J shows a displaced nucleus in a S-opsin positive cell. W: weeks of age; OS: outer segment; ONL: outer nuclear layer (photoreceptor nuclei); INL: inner nuclear layer (interneuron nuclei); IS: inner segment; M-opsin: M-opsin antibody in green; PNA: peanut agglutinin in red; DAPI: dapi counterstaining in blue; GFAP: Glial fibrillary acidic protein in green; S-opsin: short wavelength opsin in green. Scale bar in E–J represents 50 µm.</p