Comparative proteomic analysis of malformed umbilical cords from somatic cell nuclear transfer-derived piglets: implications for early postnatal death

Background: Somatic cell nuclear transfer (scNT)-derived piglets have high rates of mortality, including stillbirth and postnatal death. Here, we examined severe malformed umbilical cords (MUC), as well as other organs, from nine scNT-derived term piglets. Results: Microscopic analysis revealed complete occlusive thrombi and the absence of columnar epithelial layers in MUC (scNT-MUC) derived from scNT piglets. scNT-MUC had significantly lower expression levels of platelet endothelial cell adhesion molecule-1 (PECAM-1) and angiogenesis-related genes than umbilical cords of normal scNT piglets (scNT-N) that survived into adulthood. Endothelial cells derived from scNT-MUC migrated and formed tubules more slowly than endothelial cells from control umbilical cords or scNT-N. Proteomic analysis of scNT-MUC revealed significant down-regulation of proteins involved in the prevention of oxidative stress and the regulation of glycolysis and cell motility, while molecules involved in apoptosis were significantly up-regulated. Histomorphometric analysis revealed severe calcification in the kidneys and placenta, peliosis in the liver sinusoidal space, abnormal stromal cell proliferation in the lungs, and tubular degeneration in the kidneys in scNT piglets with MUC. Increased levels of apoptosis were also detected in organs derived from all scNT piglets with MUC. Conclusion: These results suggest that MUC contribute to fetal malformations, preterm birth and low birth weight due to underlying molecular defects that result in hypoplastic umbilical arteries and/or placental insufficiency. The results of the current study demonstrate the effects of MUC on fetal growth and organ development in scNT-derived pigs, and provide important insight into the molecular mechanisms underlying angiogenesis during umbilical cord development

Production of pigs expressing a transgene under the control of a tetracycline-inducible system.

Pigs are anatomically and physiologically closer to humans than other laboratory animals. Transgenic (TG) pigs are widely used as models of human diseases. The aim of this study was to produce pigs expressing a tetracycline (Tet)-inducible transgene. The Tet-on system was first tested in infected donor cells. Porcine fetal fibroblasts were infected with a universal doxycycline-inducible vector containing the target gene enhanced green fluorescent protein (eGFP). At 1 day after treatment with 1 µg/ml doxycycline, the fluorescence intensity of these cells was increased. Somatic cell nuclear transfer (SCNT) was then performed using these donor cells. The Tet-on system was then tested in the generated porcine SCNT-TG embryos. Of 4,951 porcine SCNT-TG embryos generated, 850 were cultured in the presence of 1 µg/ml doxycycline in vitro. All of these embryos expressed eGFP and 15 embryos developed to blastocyst stage. The remaining 4,101 embryos were transferred to thirty three surrogate pigs from which thirty eight cloned TG piglets were obtained. PCR analysis showed that the transgene was inserted into the genome of each of these piglets. Two TG fibroblast cell lines were established from these TG piglets, and these cells were used as donor cells for re-cloning. The re-cloned SCNT embryos expressed the eGFP transgene under the control of doxycycline. These data show that the expression of transgenes in cloned TG pigs can be regulated by the Tet-on/off systems

Generation of transgenic chickens expressing the human erythropoietin (<i>hEPO</i>) gene in an oviduct-specific manner: Production of transgenic chicken eggs containing human erythropoietin in egg whites

<div><p>The transgenic chicken has been considered as a prospective bioreactor for large-scale production of costly pharmaceutical proteins. In the present study, we report successful generation of transgenic hens that lay eggs containing a high concentration of human erythropoietin (hEPO) in the ovalbumin. Using a feline immunodeficiency virus (FIV)-based pseudotyped lentivirus vector enveloped with G glycoproteins of the vesicular stomatitis virus, the replication-defective vector virus carrying the <i>hEPO</i> gene under the control of the chicken ovalbumin promoter was microinjected to the subgerminal cavity of freshly laid chicken eggs (stage X). Stable germline transmission of the <i>hEPO</i> transgene to the G₁ progeny, which were non-mosaic and hemizygous for the <i>hEPO</i> gene under the ovalbumin promoter, was confirmed by mating of a G₀ rooster with non-transgenic hens. Quantitative analysis of hEPO in the egg whites and in the blood samples taken from G₁ transgenic chickens showed 4,810 ~ 6,600 IU/ml (40.1 ~ 55.0 μg/ml) and almost no detectable concentration, respectively, indicating tightly regulated oviduct-specific expression of the <i>hEPO</i> transgene. In terms of biological activity, there was no difference between the recombinant hEPO contained in the transgenic egg white and the commercially available counterpart, <i>in vitro</i>. We suggest that these results imply an important step toward efficient production of human cytokines from a transgenic animal bioreactor.</p></div

Analysis of the glycosylation pattern of egg white-hEPO derived from transgenic eggs.

<p><b>(A)</b> Intact samples treated with no enzyme. <b>(B)</b> Samples treated with PNGase F to remove <i>N</i>-linked carbohydrates. <b>(C)</b> Samples treated with <i>O</i>-glycosidase and neuraminidase to remove <i>O</i>-linked carbohydrates. <b>(D)</b> Samples treated with a combination of PNGase F, <i>O</i>-glycosidase and neuraminidase to remove all carbohydrate chains. ‘1’ and ‘2’ under each uppercase alphabetical letter indicate ‘CHO cell-derived control hEPO’ and ‘egg white-hEPO’, respectively.</p

Generation of transgenic chickens expressing the human erythropoietin (<i>hEPO</i>) gene in an oviduct-specific manner: Production of transgenic chicken eggs containing human erythropoietin in egg whites - Fig 1

<p><b>(A) Structure of the FIV-Ov19-hEPO provirus.</b> CMV/ΔLTR, a hybrid CMV promoter fused to the FIV long terminal repeat devoid of U3 region; Gag, cis-acting sequence helping efficient packaging of virus transcripts; RRE, coding sequence for the rev response element involved in packaging of the virus particles; cPPT, central polypurine track involved in integration of proviruses into the host cell genome; Ov19p, ovalbumin promoter, hEPO, human erythropoietin gene cDNA; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element sequence; ΔLTR, long terminal repeat with deletion of the U3 region to prevent replication-competent virus production after integration into the host cell genome. The approximate position of the probe for Southern blotting is shown just above the <i>hEPO</i> gene. Selected restriction enzyme sites in the provirus sequence are also indicated. In this construct, expression of the <i>hEPO</i> gene is driven by the ovalbumin promoter. Drawing is not to scale. <b>(B & C) PCR analysis of G</b>_<b>o</b> <b>(B) and G</b>_<b>1</b> <b>(C) transgenics.</b> lane P, plasmid pFIV-Ov19-hEPO; lane N, non-transgenic control chicken, lane Ov19-1, a founder transgenic rooster of G₀ generation; lanes marked with Ov19-1-1 ~ Ov19-1-4, four transgenic G₁ generation chickens sired by a transgenic G₀ rooster. <b>(D & E) Southern blot analysis of G</b>_<b>1</b> <b>transgenic chickens.</b> Genomic DNA of chickens was double-digested with <i>Spe</i> I and <i>Sbf</i> I (D) or single-digested with <i>Spe</i> I (E), then hybridized with the hEPO gene probe. Lane M, molecular size markers; the remaining lanes are same as indicated in (B & C).</p

The biological activity of egg white-hEPO.

<p>The proliferation of TF-1 cells cultured in the presence of various concentrations of hEPO derived from the eggs laid by a transgenic hen (chicken ID #Ov19-1-3, open circle,) was evaluated as described in the ‘‘Materials and Methods” section. Compared with a commercially available recombinant (closed circle), no significant difference in biological activity was determined by analysis of variance using the general linear model (GLM) procedure in the Statistical Analysis System (SAS).</p