Psoriasiform skin disease in transgenic pigs with high-copy ectopic expression of human integrins α2 and β1

Psoriasis is a complex human-specific disease characterized by perturbed keratinocyte proliferation and a pro-inflammatory environment in the skin. Porcine skin architecture and immunity are very similar to that in humans, rendering the pig a suitable animal model for studying the biology and treatment of psoriasis. Expression of integrins, which is normally confined to the basal layer of the epidermis, is maintained in suprabasal keratinocytes in psoriatic skin, modulating proliferation and differentiation as well as leukocyte infiltration. Here, we generated minipigs co-expressing integrins α2 and β1 in suprabasal epidermal layers. Integrin-transgenic minipigs born into the project displayed skin phenotypes that correlated with the number of inserted transgenes. Molecular analyses were in good concordance with histological observations of psoriatic hallmarks, including hypogranulosis and T-lymphocyte infiltration. These findings mark the first creation of minipigs with a psoriasiform phenotype resembling human psoriasis and demonstrate that integrin signaling plays a key role in psoriasis pathology

Efficient Sleeping Beauty DNA Transposition From DNA Minicircles

DNA transposon-based vectors have emerged as new potential delivery tools in therapeutic gene transfer. Such vectors are now showing promise in hematopoietic stem cells and primary human T cells, and clinical trials with transposon-engineered cells are on the way. However, the use of plasmid DNA as a carrier of the vector raises safety concerns due to the undesirable administration of bacterial sequences. To optimize vectors based on the Sleeping Beauty (SB) DNA transposon for clinical use, we examine here SB transposition from DNA minicircles (MCs) devoid of the bacterial plasmid backbone. Potent DNA transposition, directed by the hyperactive SB100X transposase, is demonstrated from MC donors, and the stable transfection rate is significantly enhanced by expressing the SB100X transposase from MCs. The stable transfection rate is inversely related to the size of circular donor, suggesting that a MC-based SB transposition system benefits primarily from an increased cellular uptake and/or enhanced expression which can be observed with DNA MCs. DNA transposon and transposase MCs are easily produced, are favorable in size, do not carry irrelevant DNA, and are robust substrates for DNA transposition. In accordance, DNA MCs should become a standard source of DNA transposons not only in therapeutic settings but also in the daily use of the SB system

The Impact of cHS4 Insulators on DNA Transposon Vector Mobilization and Silencing in Retinal Pigment Epithelium Cells

<div><p>DNA transposons have become important vectors for efficient non-viral integration of transgenes into genomic DNA. The <em>Sleeping Beauty</em> (SB), <em>piggyBac</em> (PB), and <em>Tol2</em> transposable elements have distinct biological properties and currently represent the most promising transposon systems for animal transgenesis and gene therapy. A potential obstacle, however, for persistent function of integrating vectors is transcriptional repression of the element and its genetic cargo. In this study we analyze the insulating effect of the 1.2-kb 5′-HS4 chicken β-globin (cHS4) insulator element in the context of SB, PB, and <em>Tol2</em> transposon vectors. By examining transgene expression from genomically inserted transposon vectors encoding a marker gene driven by a silencing-prone promoter, we detect variable levels of transcriptional silencing for the three transposon systems in retinal pigment epithelium cells. Notably, the PB system seems less vulnerable to silencing. Incorporation of cHS4 insulator sequences into the transposon vectors results in 2.2-fold and 1.5-fold increased transgene expression levels for insulated SB and PB vectors, respectively, but an improved persistency of expression was not obtained for insulated transgenes. Colony formation assays and quantitative excision assays unveil enhanced SB transposition efficiencies by the inclusion of the cHS4 element, resulting in a significant increase in the stable transfection rate for insulated SB transposon vectors in human cell lines. Our findings reveal a positive impact of cHS4 insulator inclusion for SB and PB vectors in terms of increased transgene expression levels and improved SB stable transfection rates, but also the lack of a long-term protective effect of the cHS4 insulator against progressive transgene silencing in retinal pigment epithelium cells.</p> </div

Insulation of SB, PB, and Tol2 transposon vectors in ARPE-19 cells.

<p>(<b>A</b>) Percentage of retained median fluorescence intensity (MFI) for stably transfected ARPE-19 clones carrying insulated transposon vectors. Measurements were obtained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048421#pone-0048421-g002" target="_blank">Figure 2a</a>. (<b>B</b>) Comparison of mean MFI levels for insulated and uninsulated clones. Stably transfected ARPE-19 cell clones were grown for 8 weeks in the absence of selection, and eGFP expression levels were measured by flow cytometry at day 0 and day 56 of passage. (<b>C</b>) Comparison of mean MFI levels for insulated and uninsulated clones carrying 1-3 transposon insertions. (<b>D</b>) Comparison of mean MFI levels for insulated and uninsulated clones carrying 9 or more transposon insertions.</p

Transposition of SB, PB, and <i>Tol2</i> transposon vectors in ARPE-19 cells.

<p>(<b>A</b>) Schematic representation of pSBT/RGIP, pPBT/RGIP, and pTol2T/RGIP vectors. IR, inverted repeat; RSV, Rous sarcoma virus promoter; eGFP, enhanced green fluorescent protein; IRES, internal ribosome entry site; puro, puromycin resistance gene; pA, polyadenylation site. (<b>B</b>) Stable transfection rates of SB, PB, and <i>Tol2</i> transposon vectors in ARPE-19 cells. 0.125 pmol of pSBT/RGIP, pPBT/RGIP, and pTol2T/RGIP plasmid were cotransfected together with 0.02 pmol pcDNA3.1D/V5.TOPO plasmid (empty vector) or 0.02 pmol helper plasmid expressing either <i>SB100X</i> transposase, <i>iPB</i> transposase, or <i>Tol2</i> transposase. The pcDNA3.1D/V5.TOPO plasmid was also included as non-specific DNA to ensure that the total amount of DNA was 1 µg in each transfection. After 8 days of selection, puromycin resistant colonies were stained and counted. Mean ± SEM values are shown (N = 3). P values listed above the brackets were obtained by student's t-tests. (<b>C</b>) Transposon copy number of stably transfected ARPE-19 clones. Genomic DNA from ARPE-19 cell clones carrying SBT/RGIP, PBT/RGIP, or Tol2T/RGIP transposons was purified and examined by Southern blot analysis to determine the transposon copy number. A representative Southern blot is shown.</p

Silencing of SB, PB, and <i>Tol2</i> transposon-based vectors in ARPE-19 cells.

<p>(<b>A</b>) Percentage of retained median fluorescence intensity (MFI) for stably transfected ARPE-19 clones. ARPE-19 cells were transfected with pSBT/RGIP, pPBT/RGIP, or pTol2T/RGIP together with a transposase expressing plasmid, and selected for puromycin resistance. Individual clones were expanded and then passaged for 8 weeks in the absence of selection. Their eGFP expression level was determined at day 0 and day 56 of passage, and their percentage of retained MFI was calculated. (<b>B</b>) Percentage of retained median fluorescence intensity (MFI) for stably transfected ARPE-19 clones containing 1–3 transposon insertions. (<b>C</b>) Percentage of retained median fluorescence intensity (MFI) for stably transfected ARPE-19 clones containing 9 or more transposon insertions. (<b>D</b>) Reactivation of eGFP expression by TSA treatment. A subset of silenced ARPE-19 cell clones was grown in the presence of the deacetylase inhibitor Trichostatin A (TSA). The clones were treated 24 hours before analysis of eGFP expression by flow cytometry.</p

<i>In silico</i> analysis results.

<p>The <i>in silico</i> analysis results of the wild-type pig ESE (+6C, CAAACAA) and the <i>Smn2</i>-like mutation (+6T, TAAACAA).</p

Genomic structure of SMN1 genes in humans, pigs and mice.

<p><b>A</b>) <i>SMN1</i> pre-mRNA transcripts expressed in humans, pigs and mice. Exons included in transcripts are numbered according to historical nomenclature. Presence of pseudoexons in processed introns are indicated in dashed outline and coloured according to the species expressing transcripts including these exons. Introns are drawn to scale and indicated as lines, exons are not drawn to scale and are indicated as boxes. Start-codon is indicated by ATG and stop-codon by TAA. <b>B</b>) The start-sequence of intron 7 in humans, pigs and mice. Bases that differ from the human sequence are indicated in bold underline. The location of the human ISS is indicated in shade.</p

Splicing analysis of SMN minigenes.

<p><b>A</b>) Summary of the SMN minigene and the mutations introduced in the different constructs. The hnRNP A1 binding sites within ISS-N1 have been indicated in dashed outline. Capitals indicate exonic bases. Bold underlined bases are bases that differ between humans and pigs. Bold italic bases in blue are mutations introduced. The +6C>T mutation is indicated in bold italic red. Dots within the sequence indicate a gap spanning multiple bases. Construct numbers correspond to lane numbers in B. <b>B</b>) RT-PCR results following transfection of Yucatan fibroblasts with minigene constructs. Inclusion expressed as a percentage is indicated in the barplot, error bars indicate standard error of mean, n = 3. Lane numbers correspond to construct numbers in A.</p