Untersuchungen zur epigenetischen Reprogrammierung nach somatischem Zellkerntransfer beim Rind mit Hilfe eines Oct4-EGFP-Reportergenkonstruktes

Die normale Entwicklung von Embryonen nach somatischem Zellkerntransfer („somatic cell nuclear transfer“, SCNT) hängt unter anderem von der erfolgreichen Reprogrammierung und Aktivierung von Schlüsselgenen ab. Ein Beispiel ist das Gen für den Transkriptionsfaktor Oct4, der im frühen Embryo nachweisbar und als Marker für pluripotente Zellen gilt. Die in klonierten Mausembryonen häufig beobachtete abnormale Expression von Oct4 wird als eine mögliche Ursache für die hohen Verluste und schweren Fehlentwicklungen nach Kerntransfer diskutiert. Beim Rind liegt im Vergleich zur Maus und anderen Spezies die Erfolgsrate am höchsten. Daher ist die Untersuchung der Reprogrammierung von OCT4 nach SCNT in der frühen Embryogenese beim Rind von besonderem Interesse. Um Fragen der epigenetischen Reprogrammierung des Rindergenoms und der Rolle von OCT4 nach SCNT nachzugehen, wurden bovine fetale Fibroblasten stabil mit einem Oct4-EGFP-Reportergenkonstrukt transfiziert. In den unilokulär stabil transfizierten Zellen mit unauffälligem weiblichen Karyotyp war in Analogie zur Inaktivität des endogenen OCT4-Gens in differenzierten Zellklonen keine EGFP-Fluoreszenz nachweisbar. Das Anschalten der Oct4-EGFP-Expression nach SCNT entsprach weitgehend der nach in vitro Fertilisation beobachteten Aktivierung des endogenen OCT4-Gens. In SCNT-Embryonen mit weniger als neun Zellkernen wurde keine EGFP-Fluoreszenz nachgewiesen. In allen Embryonen mit mindestens 17 Zellkernen war das Oct4-EGFP-Reportergenkonstrukt aktiv, was darauf hindeutet, dass Blastomeren nach der vierten Zellteilung den Oct4-Promotor aktivierten. Mittels konfokaler Laser Scanning Mikroskopie wurde an zentralen optischen Schnitten die Intensität der EGFP-Fluoreszenz jedes Embryos gemessen. Im Vergleich mit Tag 4 SCNT-Embryonen war die EGFP-Fluoreszenz in Tag 6 Embryonen deutlich stärker mit erheblichen Unterschieden in der Expressionshöhe zwischen einzelnen Embryonen. Dabei zeigten Embryonen mit einer niedrigeren EGFP-Fluoreszenz im Vergleich zu Embryonen mit stärkerer EGFP-Fluoreszenz einen erheblich höheren Anteil an Zellkernuntergängen (kondensierte und fragmentierte Zellkerne). 34 Tage nach dem Transfer von EGFP-exprimierenden Embryonen auf Empfängertiere wurden drei lebende und morphologisch unauffällige Feten gewonnen. In Fibroblasten, die aus diesen Feten isoliert wurden, war das Reportergenkonstrukt, analog zur normalen Inaktivierung des endogenen OCT4-Gens in differenzierten Zellen, inaktiviert. In SCNT-Embryonen aus den Oct4-EGFP-transgenen Fibroblasten dieser zweiten Generation („second round“ SCNT) wurde das Reportergenkonstrukt erneut regelmäßig aktiviert wie in den SCNT-Embryonen vom Ausgangszellklon („first round“ SCNT). Die Herstellung stabil transfizierter boviner fetaler Fibroblasten mit einer unilokulären Integration des Oct4-EGFP-Reportergenkonstruktes stellt eine wichtige Basis für ein breites Spektrum experimenteller Ansätze zur Aufklärung grundlegender Mechanismen nach Kerntransfer beim Rind dar

Ubiquitous LEA29Y Expression Blocks T Cell Co-Stimulation but Permits Sexual Reproduction in Genetically Modified Pigs

We have successfully established and characterized a genetically modified pig line with ubiquitous expression of LEA29Y, a human CTLA4-Ig derivate. LEA29Y binds human B7.1/CD80 and B7.2/CD86 with high affinity and is thus a potent inhibitor of T cell co-stimulation via this pathway. We have characterized the expression pattern and the biological function of the transgene as well as its impact on the porcine immune system and have evaluated the potential of these transgenic pigs to propagate via assisted breeding methods. The analysis of LEA29Y expression in serum and multiple organs of CAG-LEA transgenic pigs revealed that these animals produce a biologically active transgenic product at a considerable level. They present with an immune system affected by transgene expression, but can be maintained until sexual maturity and propagated by assisted reproduction techniques. Based on previous experience with pancreatic islets expressing LEA29Y, tissues from CAG-LEA29Y transgenic pigs should be protected against rejection by human T cells. Furthermore, their immune-compromised phenotype makes CAG-LEA29Y transgenic pigs an interesting large animal model for testing human cell therapies and will provide an important tool for further clarifying the LEA29Y mode of action

Functional evaluation of hTM, CD46 and HLA-E transgenes in vitro and in pig leg xenoperfusion experiments

Background Besides α1,3 galactosyltransferase (Gal) gene knockout several transgene combinations to prevent pig-to-human xenograft rejection are being investigated. hCD46/HLA-E double transgenic pigs were tested for prevention of xenograft rejection in an ex vivo pig-to-human xenoperfusion model. In addition, expression of human thrombomodulin (hTM-) on wild-type and/or multi-transgenic (GalTKO/hCD46) background was evaluated to overcome pig-to-human coagulation incompatibility. Methods hCD46/HLA-E double transgenic as well as wild-type pig forelimbs were ex vivo perfused with whole, heparinized human blood and autologous blood, respectively. Blood samples were analyzed for production of porcine and/or human inflammatory cytokines. Biopsy samples were examined for deposition of complement proteins as well as E-selectin and VCAM-1 expression. Serial blood cell counts were performed to analyze changes in human blood cell populations. In vitro, PAEC were analyzed for ASGR1 mediated human platelet phagocytosis. In addition, a biochemical assay was performed using hTM-only and multi-transgenic (GalTKO/hCD46/hTM) pig aortic endothelial cells (PAEC) to evaluate the ability of hTM to generate activated protein C (APC). Subsequently, the anti-coagulant properties of hTM were tested in a microcarrier based coagulation assay with PAEC and human whole blood. Results No hyperacute rejection was seen in the ex vivo perfusion model. Extremity perfusions lasted for up to 12 h without increase of vascular resistance and had to be terminated due to continuous small blood losses. Plasma levels of porcine IL1β (P < 0.0001), and IL-8 (P = 0.019) as well as human C3a, C5a and soluble C5b-9 were significantly (P < 0.05–<0.0001) lower in blood perfused through hCD46/HLA-E transgenic as compared to wild-type limbs. C3b/c, C4b/c, and C6 deposition as well as E-selectin and VCAM-1 expression were significantly (P < 0.0001) higher in tissue of wild-type as compared to transgenic limbs. Preliminary immunofluorescence staining results showed that the expression of hCD46/HLA-E is associated with a reduction of NK cell tissue infiltration (P < 0.05). A rapid decrease of platelets was observed in all xenoperfusions. In vitro findings showed that PAEC express ASGR1 and suggest that this molecule is involved in human platelet phagocytosis. In vitro, we found that the amount of APC in the supernatant of hTM transgenic cells increased significantly (P < 0.0001) with protein C concentration in a dose-dependent manner as compared to control PAEC lacking hTM, where the turnover of the protein C remained at the basal level for all of the examined concentration. In further experiments, hTM also showed the ability to prevent blood coagulation by three- to four-fold increased (P < 0.001) clotting time as compared to wild-type PAEC. The formation of TAT complexes was significantly lower when hTM-transgenic cells (P < 0.0001) were used as compared to wild-type cells. Conclusions Transgenic hCD46/HLA-E expression clearly reduced humoral xenoresponses since the terminal pathway of complement, endothelial cell activation, inflammatory cytokine production and NK-cell tissue infiltration were all down-regulated. We also found ASGR1 expression on the vascular endothelium of pigs, and this molecule may thus be involved in binding and phagocytosis of human platelets during pig-to-human xenotransplantation. In addition, use of the hTM transgene has the potential to overcome coagulation incompatibilities in pig-to-human xenotransplantation

Nanopollution: the invisibile fog of future wars

The wars that ended the twentieth century and launched the twenty-first brought a mysterious, unexpected side effect. Some postwar pathologies, such as soldiers' malformed offspring and the illnesses of peacekeepers deployed to former battlefields, raise crucial questions. Other than coming up with a name for the collection of symptoms experienced by veterans of the first two Gulf Wars and the war in the Balkans, doctors have little understanding of Gulf War syndrome's causes. The solution to these medical mysteries may be found in a new word: nanopathology. Dust at the nanoscale can elude physiological barriers and easily enter the bloodstream, where it is very likely to reach all internal organs and tissues. Nano-war is a real risk that is already with us, even if governments seem to be unaware of it. The obvious (but overly simplistic) solution is to stop making war. The second best solution is to stop pretending nanodust either doesn't exist or is harmless

Biological evaluation of transgenic founder animals.

<p>(A) Binding studies of LEA29Y to the CD80/CD86-positive porcine B cell line L23. Cells were incubated with serial dilutions of sera from the three founder animals as well as from wild-type controls. Binding of LEA29Y was assessed by using a goat anti-human IgG-FITC antibody. Labeled cells were analyzed by flow cytometry. The results are expressed as the mean fluorescence intensity. (B) Inhibition of human anti-pig T cell proliferation by serum from LEA29Y-tg pigs. 10⁵ human PBMC were stimulated with 2 x 10³ irradiated L23 cells. Cultivation was performed in the presence of sera taken from transgenic and wild-type control pigs. Proliferation was determined after 5 d by [³H]-TdR incorporation (ccpm, counts/min). The results are expressed as the mean ± SD of triplicate cultures.</p

Lymph node histology in transgenic pigs.

<p>Developmental stage of lymph nodes of a 6-month-old CAG-LEA transgenic pig (B, E) in comparison with WT porcine lymph nodes; the age of the animals was 19 days (A, D) and 6 months (C, F). Immunohistochemistry using the PPT3 anti-CD3 antibody demonstrates nearly T cell-free follicles (Fo) near the trabecules (T) in all samples. Follicles in samples of 6-month-old WT animals were generally larger than in transgenic or young animals (compare C versus A and B). Follicles of 6-month-old WT porcine lymph nodes showed a distinct reaction center (F) with heavily proliferating centroblasts in the dark zone (DZ) and less proliferating cells in the pale zone (PZ)–comparing strong and weak Ki67 immunopositivity (proliferation marker) in DZ and PZ, respectively. In contrast, marked proliferation of lymphocytes could be detected in either the follicles of transgenic (E) nor 19-day-old WT animals (D). The amount of proliferating T cells did not seem to differ between the samples. Visualization of the immunoreaction diaminobenzidine—horseradish peroxidase (positive staining = brown), counterstaining hematoxylin, scale bar = 200 μm.</p

Genetic distance between mammalian CTLA4, B7.1/CD80 and B7.2/CD86.

<p>The percentage of amino acid identities between pairwise comparisons are shown as a matrix for CTLA4 (A), CD80 (B) and CD86 (C). The distribution of pairwise identities was calculated in segments of 4% (D) and illustrates higher amino acid conservation in CTLA4 compared to CD80 and CD86.</p

Localization of LEA29Y expression in F1 generation.

<p>Immunohistochemistry was performed using an antibody specific for the human IgG tail of LEA29Y. (A) LEA29Y was predominantly detected in endothelial cells including capillaries and interstitia, as well as in organ- and tissue-specific cell types such as pulmonary alveolar cells, exocrine pancreas cells, bile duct cells of the liver, or the stratum spinosum cell layer of the skin. Endocrine cells of the Langerhans islet (indicated by an arrow) as well as the thyroid gland exhibited strong staining. Additionally, intravascular serum stained positive for LEA29Y. (B) No immune staining could be detected in WT control tissue. Chromogen: DAB; nuclear staining: hemalum; scale bar = 200 μm. Inset in pancreas: Giemsa stain of the corresponding pancreas region to demonstrate Langerhans islet. Lymph node: L. tracheobronchialis medialis.</p