CEP290 protein analysis in <i>Cep290</i> mouse models.

<p>Representative CEP290 immunodetection in P150 retinas from the three different mouse models. A CEP290-Flag construct was expressed in HEK293-T cells and used as a positive control. Tubulin was used for normalization.</p

Generation of the <i>Cep290</i> humanized mouse models.

<p>(A) Structure of the <i>CEP290</i> gene in human and mouse (drawn to scale). (B) Two mouse models were generated by introducing human exons 26 and 27 and intron 26, either with or without the LCA-causing mutation (depicted with *), to the mouse <i>Cep290</i> gene. Human and mouse loci, targeting vector and recombinant locus are depicted. Arrows and letters indicate the position of the oligonucleotides (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079369#pone.0079369.s001" target="_blank">Table S1</a>). </p

Morphological and CEP290 immunolocalization analyses in P150 mouse retinas.

<p>Immunodetection of CEP290 and acetylated tubulin in retinal sections from <i>Cep290</i>^<i>wt/wt</i>, <i>Cep290</i>^{<i>hum/hum</i>} and <i>Cep290</i>^{<i>lca/lca</i>} mice, at P150. Images were taken at 200X (A) and 400x (B) magnifications. CEP290 (green) is located in the connecting cilium (CC) and remains unaltered in the humanized models. Nuclei were marked with DAPI (blue) and CC with acetylated tubulin (red). GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; PhL: photoreceptor layer; RPE: retinal pigment epithelium; IS: inner segment; CC: connecting cilium; OS: outer segment.</p

Analysis of the <i>CEP290</i> cryptic exons.

<p>(A) Expected transcripts for each model. (B) Exon Y is expressed in all tissues in either <i>Cep290</i>^{<i>hum/hum</i>} and <i>Cep290</i>^{<i>lca/lca</i>} mice. (C) Only in the <i>Cep290</i>^{<i>lca/lca</i>} model, exon X is expressed, either with exon Y or without, at very low levels. The arrow indicates the band that contains both cryptic exons (exon X and exon Y) in the same transcript. <i>Cep290</i>^lca/lca PCR products were run longer to clearly separate the two different bands. (D) Schematic representation of the different transcripts containing cryptic exons found in the three mouse models. Mouse exons and introns are depicted in black, while human exons and introns are represented in blue. Cryptic exons X and Y are shown in grey and red, respectively. Arrows and letters indicate the position of the oligonucleotides in the cryptic exons. Semi-quantification was performed using ImageJ software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079369#B21" target="_blank">21</a>]. MQ: H₂O (negative control); F: human LCA fibroblasts; B: Brain; K: Kidney; R: Retina; Lu: Lung; S: Spleen; T: Testis and Li: Liver.</p

Transcriptional characterisation of <i>Cep290</i> in humanized mouse models.

<p>(A-B) <i>Cep290</i> expression levels in various mouse tissues were assessed by RT-PCR. Regions containing murine exons 10 to 13 (A) and 24 to 27 (B) were analyzed. No differences were observed. (C) Amplification using human primers was also assessed in the three models. Only humanized models showed amplification. (D) Actin was used for normalization and comparison among tissues and models. MQ: H₂O (negative control); B: Brain; K: Kidney; R: Retina; Lu: Lung; S: Spleen; T: Testis and Li: Liver.</p

Analysis of the cryptic exons X and Y in human retina and fibroblast cell lines.

<p>The expression of the <i>CEP290</i> human exons 26 and 27, and the cryptic exons Y and exon X were assessed in tissues derived from both mice and humans. Actin was used for normalization. </p

<i>In situ</i> hybridization of genes encoding DUB enzymes on retinal cryosections.

<p>Sections from wild-type C57BL/6J mouse retinas were hybridized using digoxigenin-labelled antisense riboprobes. Their corresponding sense riboprobes (negative controls) stained for the same length of time (lower panels in each row) are in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150364#pone.0150364.s001" target="_blank">S1 Fig</a>. The antisense <i>Rhodopsin</i> probe, which strongly labels the inner photoreceptor segment, was used as a positive control for the assay. <b>RPE</b>- Retinal pigmented epithelium; <b>Phr</b>- Photoreceptor cell layer; <b>ONL</b>- Outer nuclear layer; <b>OPL</b>. Outer plexiform layer; <b>INL</b>- Inner nuclear layer, <b>IPL</b>- Inner plexiform layer; <b>GCL</b>- Ganglion cell layer.</p

Counts of classified DUB homologs.

Heatmap representing the number of classified genes in each analyzed genome. Increasing intensity reflects increasing number of genes. Only orthologs marked with black dots in Fig 4 are considered. Acropora digitifera USP homologs, excluded from the phylogenetic classification, are marked as not analyzed (NA).</p

Relative expression levels of the five subfamilies of DUB enzymes.

Gene expression values are the average of three independent samples (measured in three replicates), each sample contained retinas from three individuals. The expression levels are obtained as a ratio with Gapdh expression (used for normalization) per 104. The Z-score has been calculated for the whole set of genes per each sample, and mean and standard deviation has been obtained, so that the results can be directly compared among them. Negative values indicate when genes are expressed below the global mean of the gene expression obtained in the analysis, and positive values when genes are more highly expressed. To simplify the comparison, the graph starts at the negative values, being 0 the mean value of gene expression for the whole set of genes (87 in total) in each sample. JAMM- JAB1/MPN/MOV34 motif proteases; MJD- Machado-Joseph Disease protein domain proteases; UCH- Ubiquitin C-Terminal Hydrolases; OTU- Ovarian Tumor proteases; USP- Ubiquitin-Specific Proteases.</p

Rubisco supercluster (cluster id: 410) actual and possible matches.

Rubisco supercluster (cluster id: 410) actual and possible matches.</p