Functional analysis of bicistronic SB transposon vectors.

<p>(<b>A</b>) Schematic representation of bicistronic SB-transposon vectors carrying the hITGB1 gene driven by CMV and INV promoters, respectively. LIR and RIR indicate the SB transposon left and right inverted repeats, respectively; SV40 pA and BGH pA, indicate the Simian virus 40 and bovine growth hormone polyadenylation sites, respectively. (<b>B</b>) Stable transfection rate in NIH3T3 cells co-transfected with equimolar amounts of pT2/SV40-neo or pT2/CMV-hITGB1. SV40-neo in conjunction with pCMV-mSB or pCMV-SB100X. pUC19 was included as stuffer in some transfections to ensure that equal amounts of DNA were transfected. G418-resistant colonies were counted after 14 days selection. (<b>C</b>) Western blot analysis using a mouse anti-human β1 integrin mAb (610467) on cellular protein extracts from naïve NIH3T3 cells or NIH3T3 cells stably transfected with pT2/CMV-hITGB1. SV40-neo (top panel). Top band is mature β1 integrin, lower band pre-β1 integrin. Detection of ACTB with an anti-β-actin mAb served as loading control (lower panel). (<b>D</b>) Transposase titration assay in Göttingen primary fibroblasts co-transfected with 0–100 ng pCMV-SB100X and 1.9 µg pT2/CMV-hITGB1. SV40-neo. Resistant colonies were counted after 14 days of selection with G418. (<b>E</b>) Quantitative RT-PCR on total mRNA extracted from naïve or pT2/CMV-hITGB1. SV40-neo stably transfected Göttingen primary fibroblasts. Human β1 integrin specific exon-exon primers were utilized to detect transcripts derived from the integrated transposon. The mRNA level was normalized to the level of endogenous β-actin mRNA. (<b>F</b>) Transposition assay in HaCaT cells co-transfected with 1.9 µg pT2/SV40-neo or pT2/INV-hITGB1. SV40-neo in conjunction with 100 ng pCMV-mSB or pCMV-SB100X. Resistant colonies were counted following 14 days of G418 selection. (<b>G</b>) Immunostaining of permeabilized naïve (a) or pT2/INV-hITGB1. SV40-neo-transfected (b) HaCaT cells. A mouse anti-human β1 integrin mAb (P5D2) was used to identify β1 integrin protein derived from the transposon expression cassette. Data are presented as mean values ± standard deviations.</p

Ectopic expression of the β1 integrin in the transgenic pigs.

<p>(<b>A</b>) Total RNA obtained from non-transgenic (#C1) and hITGB1 transgenic pig skin-biopsies were analyzed by human β1 integrin specific qRT-PCR, normalized to endogenous β-actin. Significant expression was detected in all transgenic pigs. Data are presented as mean values ± standard deviations. (<b>B</b>) Representative wide-field fluorescence microscopy images of permeabilized frozen cutaneous sections (from a non-transgenic pig (#2990) and hITGB1-transgenic pigs) stained with an anti-human β1 integrin mAb (P5D2). Staining of basal cells is evident in all pigs (peach arrows), but subrabasal expression is apparent in transgenic animals only (grey arrows). Basal membrane is indicated by a white dashed line and the skin surface by a grey dashed line. In b the basal membrane is located to the lower right outside the figure.</p

Increased production and secretion of IL-1α from transgenic keratinocytes.

<p>(<b>A</b>) Total RNA isolated from hITGB1-transgenic keratinocytes was used for porcine IL-1α specific qRT-PCR analysis. Expression levels of IL-1α were increased up to 6-fold in transgenic cells compared to a non-transgenic control (#2990). (<b>B</b>) qRT-PCR analysis for detection of IL-1α mRNA in hITGA2-transgenic keratinocytes. Messenger RNA levels were markedly elevated in transgenic cells compared to non-transgenic controls (#2990, #301703 and #301706). Images of gel-electrophoresed qRT-PCR products are depicted (insert); C indicates the water control. (<b>C</b>) Secretion of IL-1α measured by porcine IL-1α specific ELISA. Conditioned medium was harvested from hITGB1-transgenic keratinocytes and non-transgenic controls (#301703 and #301706) after 14 hours of incubation and used for ELISA. Secretion of IL-1α was increased up to 3-fold from transgenic cells. Data are presented as mean values ± standard deviations. Asterisks (*) indicate statistical significance compared to pig #2990 (A–B) or #301703 (C).</p

Expression and cell membrane localization of heterologous β1 integrin.

<p>(<b>A</b>) Quantitative RT-PCR for quantification of β1 integrin mRNA performed on total mRNA extracted from cultured keratinocytes derived from a non-transgenic pig (#2990) and the six hITGB1 transgenic pigs, normalized to endogenous β-actin. The expression level of human β1 integrin was proportional to genomic integration copies and only detectable in the transgenic animals. Data are presented as mean values ± standard deviations. (<b>B</b>) Western blot analysis on total cellular protein from the keratinocytes described in (A). Employing the mouse anti-human β1 integrin mAb (610467), both pre- and post-translationally processed human β1 integrin protein is detected in all transgenic pigs. ACTB detection was used as loading control. (<b>C</b>) Flow cytometry analysis of keratinocytes stained with an anti-human β1 integrin mAb (P5D2) without permeabilization (light gray bars) or with saponin permeabilization (dark gray bars). Representative histograms comparing fluorescence from pig #2990 and #3404 with and without permeabilization are depicted. In five out of the six hITGB1 transgenic keratinocytes, β1 integrin was predominately localized to the cell membrane. (<b>D</b>) Representative images from wide-field (a–b) and confocal (c–d) fluorescence microscopy of permeabilized keratinocytes (derived from the non-transgenic control (#2990) and hITGB1 transgenic pigs) stained with anti-human β1 integrin (P5D2). Detection of β1 integrin was restricted to transgenic cells with accumulation at the plasma membrane and especially at cell-cell adhesion sites.</p

Demonstration of transgenesis and lack of SB100X gene integrations by genotyping of cloned pigs produced by HMC.

<p>(<b>A</b>) Photo of the six cloned pigs at one week of age. (<b>B</b>) PCR analysis on genomic DNA extracted from cultured pig fibroblasts. Neomycin- and hITGB1-specific internal primer-pair demonstrated the presence of the transgenic cassette in the cloned pigs but not in the non-transgenic control (NT) (two upper panels). Random integration of the SB100X gene could not be detected in the cloned pigs by using SB100X-specific primers (lower panel). MG366, a cloned pig with a known SB100X insertion was used as a control; PC, plasmid control; M, 100 bp marker. (<b>C</b>) Southern blot analyses on 15 µg genomic DNA from the transgenic animals demonstrated between one and six insertions per pig. DNA was digested with EcoRV and PvuI (left panel) or with NheI and SalI (data not shown) and probed with a ³²P-labelled Neo probe. DNA from NT spiked with pT2/INV-hITGB1.SV40-neo was utilized as plasmid control. The number of insertions ranges from one to five in the six transgenic animals. An identical band appears in all lanes and is presumably non-specific. NT, non-transgenic control.</p

Erk1/2 phosphorylation in TPA-stimulated hITGB1-transgenic keratinocytes but not in hITGA2-transgenic keratinocytes.

<p>(<b>A</b>) Naïve and hITGB1- or hITGA2–expressing HaCaT cells were grown to subconfluency on uncoated or collagen I-coated plates. After two days of incubation in serum-depleted medium, one subset was stimulated with 100 ng/mL TPA for 10 min, after which all subsets were stained with a phosphor-Erk1/2 specific mouse mAb (E10). Increased levels of pERk1/2, indicative of increased Erk/MAPK activation, could be detected in stimulated β1 integrin-expressing HaCaT cells, whereas an inhibition upon TPA stimulation was apparent in α2 integrin-expressing cells. Asterisks (*) indicate statistical significance relative to naïve HaCaT cells. (<b>B</b>) TPA-induced activation of Erk1/2 phosphorylation in transgenic keratinocytes. Cultured hITGB1-transgenic keratinocytes were treated as described under (<b>A</b>). In case of pig #2408, a marked difference in phosphorylated Erk1/2 levels was detected by comparing TPA-stimulated and un-stimulated cells. Data are presented as mean values ± standard deviations.</p

Elevated c-Fos mRNA expression levels in un-stimulated transgenic pig keratinocytes.

<p>(<b>A</b>) Quantitative RT-PCR on total mRNA extracted from cultured keratinocytes derived from non-transgenic pigs (#2990 and #3632) and the six hITGB1-transgenic pigs, normalized to endogenous β-actin. A modest increment of endogenous c-Fos mRNA was detectable in four out of the six transgenic keratinocytes compared to the levels in control pigs #2990 and #3632. (<b>B</b>) Quantification of c-Fos mRNA levels in hITGA2-expressing keratinocytes was performed as described in (A). Images of gel-electrophoresed qRT-PCR products are depicted (insert); C indicates the water control. A substantial increase of c-Fos mRNA levels was recorded in the transgenic keratinocytes derived from the three hITGA2-transgenic pigs compared to the levels in control pig #2990. Data are presented as mean values ± standard deviations. Asterisks (*) indicate statistical significance compared to #2990.</p

Altered cytokine and chemokine profile in hITGB1-transgenic animals.

<p>Quantitative RT-PCR directed against ten molecular markers of skin-inflammation performed on total RNA extracted from punch biopsies taken from the non-transgenic pig #301702 and the hITGB1-transgenic pigs #2410 and #3402 at age 14 months, normalized to endogenous GAPDH. Data are technical triplicates and presented as mean values ± standard deviations. CCL5, CCL20, and CCL27 (Chemokine (C-C motif) ligand 5, 20, and 27, respectively); CXCL10 (chemokine (C-X-C motif) ligand 10); IL-8 and IL-1β (interleukins 8 and 1β); TNF-α (tumor necrosis factor alpha); GM-CSF (granulocyte-macrophage colony-stimulating factor); PCNA (proliferating cell nuclear antigen).</p

Infiltration of human IL-17A-producing T cells and CD4+ Foxp3-expressing T cells in the inflamed human skin in the SCID/skin allograft mouse model.

<p><b>A.</b> Immunohistochemistry of human CD4 (brown, top) and CD8 (brown, bottom) expression in human skin grafts from SCID beige mice 21 days after infusion or huPBMC. Photographs show representative examples. 20× magnification. <b>B.</b> Summarized data of figure A. showing mean±SEM CD4 (top) or CD8 (bottom) positive cells/mm² of n = 4 and 10 upon PBS and huPBMC infusion resp in the epidermis (white bars) and dermis (black bars). <b>C.</b> Immunohistochemistry of human IL-17A expression in human skin grafts from SCID beige mice 21 days after infusion of PBS (left) or huPBMC (right). Photographs show representative examples of n = 6 (huPBMCs) n = 3 (controls). 20× magnification. Graph shows summarized data of IL-17A positive cells/mm² following PBS or huPBMC infusion in the human skin biopsies (mean±SEM, of n = 4 and 10). <b>D.</b> Immunohistochemistry of coexpression of human CD4 (blue) and IL-17A (red, top) and CD8 (blue) and IL-17A (red, bottom) in human skin grafts from SCID beige mice 21 days after infusion of huPBMC (20× magnification). Inserts show a higher magnification (40×) of single CD4/CD8 and IL-17A staining and CD4/IL-17A or CD8/IL-17A co-staining. . Photographs show representative examples of n = 6. <b>E.</b> Immunohistochemistry of IL-17A (red) in human mastcell tryptase (brown) and granulocyte elastase (brown) in human skin biopsies from SCID beige mice 21 days after infusion of PBS (left) or huPBMC (right). Photographs show representative examples of n = 6 (huPBMCs) n = 3 (controls). 20× magnification. <b>F.</b> Immunohistochemistry of co-expression of human Foxp3 (brown) and CD4 (blue) in human skin grafts from SCID beige mice 21 days after infusion of PBS (left) or huPBMC (right) (40× magnification). Inserts show a higher magnification (63×) of single Foxp3 and CD4+ staining and Foxp3/CD4 co-staining. Photographs show representative examples of n = 6 (huPBMCs) n = 3 (controls).</p

Acanthosis and aberrant epidermal marker expression of hBD2, Elafin, K10 and K16 in the inflamed human skin of the huPBL-SCID-hu Skin allograft model.

<p><b>A.</b> Histology (H&E staining) of human skin grafts from SCID beige mice 21 days after infusion (i.p.) of huPBMC. Representative photographs are shown after PBS (left photograph) and huPBMC infusion (right photograph). Photograph shows increased epidermal thickness (acanthosis) and elongated fingerlike epidermal projections into the dermis (rete ridges) and large lymphocyte infiltration after huPBMC infusion. Abnormal presence of nuclei in the stratum corneum (parakeratosis) and infiltration of lymphocytes in the epidermis (exocytosis) (bottom-right photograph). 20×, 20×, 40× magnifications respectively. <b>B.</b> Quantitative microscopic histological analysis of the epidermal thickness (µm) of human skin grafts following infusion of PBS and huPBMC. Mean±SEM are shown for n = 3 and 6 upon PBS and huPBMC infusion resp. <b>C.</b> Immunohistochemistry of K10, K16, hBD2 and Elafin (brown) in human skin grafts from SCID beige mice 21 days after infusion (i.p.) of PBS (top panels) or huPBMC (lower panels). Photograpsh show representative examples, summarized data are given in the figures. Mean±SEM percentages of the area positive for the indicated markers is shown for n = 3 and 5–8 upon PBS and huPBMC infusion resp. 10× magnification. <b>D.</b> Ki-67 expression (brown) in the stratum basale of the epidermis. The insert shows a higher magnification. A representative example of n = 12 is shown. 20× magnification. Graphs show summarized data of Ki67+ cells/mm length of basement membrane after PBS or huPBMC infusion in human skin biopsies (mean±SEM, of n = 4 and 6, resp.).</p