TALEN design and evaluation of cutting efficiency in rat glioma C6 cells.

<p>(a) Schematic of the rat <i>Nc3r1</i> (GR) gene. Zoom on the area of the mutation pA476T in exon 3. The first nucleotide of the 476 codon is highlighted in blue. TALEN binding sites of TAL 3 are highlighted in green. Detailed sequences of TAL 6 binding sites can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088146#pone.0088146.s001" target="_blank">Table S1, File S1</a>. (<b>b</b>) T-endo1 assay results. Pooled DNA from C6 cells transfected with either Right, Left or Right and Left TALEN monomers (marked with R, L or RL, respectively) was amplified and treated with T7 endo 1 enzyme. Cut bands of 288 and 177 bp indicate TALEN activity. Mcells are mock transfected cells, GFP: GFP transfected cells were used as a positive transfection control. Intensity of the cut bands are indicated for TAL 3 and TAL 6 pairs. (<b>c</b>) TAL 3 transfected cells screening. PCR amplicons of the region around the pA76T mutation were subcloned into TOPO vector. Clones were isolated and analyzed individualy. Four point mutations and insertion.s are marked in red.</p

Injections of TAL 3 mRNA and donor plasmid DNA in rat one-cell embryos.

†<p>One pup was born dead.</p><p>Two doses of TAL 3 mRNA were used (20+20 or 10+10 ng/µl of each TALEN). The egg survival rate is shown in percentage. NHEJ indicates the number of pups that had a gene disruption event in the sequence around pA476T. The percentages were calculated within each set of TALEN mRNA amount injected.</p

Founder KI female 3.4 genotyping from subcloned PCR amplicons of tail biopsies.

<p>Wt: Wild type; DP: donor plasmid. Point mutations in the DP are indicated in red bold letters. The pA476T mutation is highlighted in blue.</p

Detection of random donor integration in rat founders.

<p>Representative Southern blot analysis of founder rat genomic DNA following <i>Hin</i>cII digestion of genomic DNA and hybridization with exon 3-derived probe. Indicated is the 3.6 kb endogenous exon 3-containing genomic fragment and additional random integrations in founder 5.4 (knockout) and 8.1 (wildtype) rats. Rats 5.5 and 6.1 are knockouts as well but presented no off-target donor integration.</p

Fo KO rat genotyping.

<p>Wt, wild type sequence. TALEN binding sites are shown in green. The pA476T mutation is highlighted in blue. Longer deletions are marked with double slash. Rats 11.4, 6.1 and 5.5 (underlined) were kept for breeding. All rats beared the wt allele of the <i>Nr3c1</i> gene. Primers used are listed in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088146#pone.0088146.s001" target="_blank">Table S2, File S1</a></b>.</p

Adoptive transfer of B cells transfers tolerance.

<p>Cells were sorted by FACS Aria <b>(sorting strategy displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119686#pone.0119686.s003" target="_blank">S3D and E Fig.</a>)</b> from the spleen of tolerant rats (>100 days after the graft) that had received a transfer of splenocytes from a previously tolerant recipient and adoptively transferred to sub-lethally irradiated recipients the day before the transplant. <b>(A)</b> B cells (CD45RA⁺, n = 3), T cells (TCR⁺, n = 4), CD8⁺ Tregs (CD8⁺CD45RC^low, n = 2), pDCs (mAb 85C7⁺ n = 3) Groups are compared with each other and to irradiated animals transferred with naive splenocytes (naive splenocytes, n = 5) by Log-rank (Mantel-Cox) Test <i>p</i> <0.05*; p<0.01**; p<0.001***. <b>(B)</b> Wild type (WT) and B cell-deficient <i>Igm</i> knockout (KO) rats were treated with AAV-FGL2 (n = 8 and 3, respectively), AAV-null (n = 5) or untreated (NT, n = 3), and analyzed for graft survival. <b>(C)</b> Splenocytes from adoptively-transferred tolerant rats were depleted in CD45RA⁺ B cells (CD45RA⁻ cells) or not (splenocytes) and transferred to new irradiated recipients. Log-rank (Mantel-Cox) Test p<0.01**. <b>(D) Left:</b> A fraction of the transferred tolerogenic CD45RA⁺ B cells was tested for inhibition of CFSE-labeled CD4⁺CD25⁻ T cell proliferation in response to allogeneic LEW.1W cDCs, pDCs (stimulator/effector ratio of 1:4) or anti-CD3 at day 6 of culture. Shaded grey: naive CD45RA⁺ B cells n = 3, black line: tolerogenic CD45RA⁺ B cells n = 4. <b>Right</b>: Representative histogram of one proliferation assay of CD4⁺CD25⁻ T cells with allogeneic pDCs and CD45RA⁺ B cells from naive (shaded grey) or splenocyte-transferred tolerant rats (black line). <b>(E)</b> Graft infiltrating cells were analyzed for the presence of CD45RA⁺ cells in graft of rats transferred with B cells, at days 100 after the graft, as compared with syngeneic grafts (n = 3).</p

Splenocytes from AAVFGL2-treated rats with long-term surviving grafts transfer donor alloantigen-specific long-term graft survival in an iterative manner.

<p>Splenocytes from long-term AAV-FGL2-treated recipients were injected <i>i</i>.<i>v</i>. into sub-lethally irradiated recipients (LEW.1A) the day before heart allotransplantation (LEW.1W). Graft survival was evaluated by palpation through the abdominal wall. Total splenocytes (1x10⁸ cells) from long-term (≥120 days) AAV-FGL2-treated rats were adoptively transferred (1^st-transferred, n = 5), and then total splenocytes (10⁸ cells) were iteratively transferred to 2^nd- (n = 6), 3rd (n = 4), 4^th (n = 3), 5^th (n = 3) and 6^th (n = 3) LEW.1A recipients receiving LEW.1W hearts. Third-party grafts were from Brown-Norway origin and adoptive transfer of splenocytes from LEW.1W-transplanted animals did not inhibit acute rejection (third party, n = 3, performed in animals that received a second adoptive transfer). Splenocytes from naive non-transplanted rats did not inhibit acute rejection (naive splenocytes, n = 5) and non-irradiated non-transferred recipients (no treatment, n = 6) also showed acute rejection. Irradiation alone without cell transfer delays graft survival but does not prevent graft from rejection (irradiated, n = 5). All groups were compared to irradiated animals transferred with naive splenocytes by Log-rank (Mantel-Cox) Test (<i>p value</i> ***<0.001).</p

Over-expression of FGL2 <i>in vivo</i> prolongs cardiac allograft survival.

<p><b>(A)</b> Cardiac graft recipients received intravenously 3x10¹²vector genomes/kg of AAV-FGL2 (▲ n = 8), or non coding AAV (▼ n = 5, 2 different experiments), and received a heterotopic transplant 30 days later. Graft survival was evaluated by palpation through the abdominal wall. Log-rank (Mantel-Cox) test ***<i>p<0</i>.<i>001</i> for AAV-FGL2 vs. AAV null controls. <b>(B)</b> Left: Relative proportion of dividing CD4⁺CD25⁻ T cells at day 6 in the presence of different concentrations of recombinant human FGL2-GST was evaluated by CFSE dilution by gating first on DAPI^- live cells and then on TCR⁺CD4⁺ cells <b>(<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119686#pone.0119686.s002" target="_blank">S2B Fig.</a>).</b> The negative control was purified rat IgG at 10 μg/ml (n = 4, ** <i>p</i><0.01). Right: Representative histogram of relative proportion of dividing CD4⁺T cell in the presence of 10μg/ml FGL2-GST protein (black line) or IgG control (grey).</p

Alloantibody production was suppressed after AAV-FGL2 treatment and adoptive transfer of splenocytes.

<p>Sera were collected from naive rats, or at the moment of rejection from rats treated with AAV-control or AAV-FGL2 (rejecting at < 30 days or > 120 days after transplantation) or receiving adoptive transfers (> 120 days after transplantation). Levels of donor-specific IgG1, IgG2a, and IgG2b antibodies were evaluated by cytofluorimetry and normalized to serum from naive rats (MFI / MFI syngeneic). Two way Anova, Bonferroni post test <i>p value</i> * <0.5; ** <0.01; ***<0.001.</p

Severe and progressive muscle changes in <i>Dmd^mdx</i> rats.

<p><i>Biceps femoris</i> muscle was sampled at 3 and 7 month-old from wild-type littermate controls (WT) and <i>Dmd^mdx</i> rats. Compared to controls (<i>A</i>), 3 month-old <i>Dmd^mdx</i> rat skeletal muscles (<i>B</i>) displayed individual fiber necrosis (open arrowhead) associated with foci of small newly regenerating centronucleated fibers (arrow). In addition, 7 month-old muscles displayed a progessive replacement of fibers by fibrosis (*) and fat (black arrowhead) tissue (<i>C</i>). The regenerative activity was assessed using a specific antibody against the Myosin Heavy Chain developmental isoform (MyoHC_Dev). No regenerative activity was observed in control rats (<i>D</i>) whereas newly regenerating fibers were numerous in <i>Dmd^mdx</i> rats (<i>E</i>) with a decrease in their number with age (<i>F</i>). Histological changes were quantified. Fiber minimal Ferret diameter was measured. A global switch towards lower sized fibers was observed in the fiber size distribution in mutated rats compared to controls at 3 and 7 month of age (<i>G–H</i>). Hemalun eosin saffron staining (<i>A–C</i>), immunolabelling of Myosin Heavy Chain developmental isoform (MyoHC_Dev) (<i>D–F</i>). Bar = 100 µm (<i>A–C</i>) and 200 µm (<i>D–F</i>).</p