The increasing focus on the pig as a biomedical model calls for studies which investigate morphological and molecular mechanisms during initial embryonic development in this species. In the pig, the paternal genome is actively demethylated in the zygote, whereas the maternal genome remains methylated. The major genome activation occurs at the four-cell stage, when prominent ribosome-synthesizing nucleoli develop in the blastomeres, allowing for trophectoderm and inner cell mass (ICM) differentiation. Unlike in mice, the pluripotency gene OCT4 is initially expressed in both compartments. The ICM differentiates into epiblast and hypoblast approximately at the time of hatching from the zona pellucida, and subsequently the loss of the Rauber's layer results in an uncovered epiblast establishing the embryonic disc again in contrast to mice. This particular and protracted ICM/epiblast biology may contribute to the lack of success in culturing porcine embryonic stem cells. The embryonic disc subsequently becomes polarized by a posterior thickening, which includes ingression of the first extra-embryonic mesoderm. Thereafter, the primitive streak forms and gastrulation results in formation of the somatic germ layers and germline, i.e. the primordial germ cells. The latter remain pluripotent for a period and may be isolated and cultured as embryonic germ cells in vitro.

BackgroundEpigenetic reprogramming is critical for genome regulation during germ line development. Genome-wide demethylation in mouse primordial germ cells (PGC) is a unique reprogramming event essential for erasing epigenetic memory and preventing the transmission of epimutations to the next generation. In addition to DNA demethylation, PGC are subject to a major reprogramming of histone marks, and many of these changes are concurrent with a cell cycle arrest in the G2 phase. There is limited information on how well conserved these events are in mammals. Here we report on the dynamic reprogramming of DNA methylation at CpGs of imprinted loci and DNA repeats, and the global changes in H3K27me3 and H3K9me2 in the developing germ line of the domestic pig.ResultsOur results show loss of DNA methylation in PGC colonizing the genital ridges. Analysis of IGF2-H19 regulatory region showed a gradual demethylation between E22-E42. In contrast, DMR2 of IGF2R was already demethylated in male PGC by E22. In females, IGF2R demethylation was delayed until E29-31, and was de novo methylated by E42. DNA repeats were gradually demethylated from E25 to E29-31, and became de novo methylated by E42. Analysis of histone marks showed strong H3K27me3 staining in migratory PGC between E15 and E21. In contrast, H3K9me2 signal was low in PGC by E15 and completely erased by E21. Cell cycle analysis of gonadal PGC (E22-31) showed a typical pattern of cycling cells, however, migrating PGC (E17) showed an increased proportion of cells in G2.ConclusionsOur study demonstrates that epigenetic reprogramming occurs in pig migratory and gonadal PGC, and establishes the window of time for the occurrence of these events. Reprogramming of histone H3K9me2 and H3K27me3 detected between E15-E21 precedes the dynamic DNA demethylation at imprinted loci and DNA repeats between E22-E42. Our findings demonstrate that major epigenetic reprogramming in the pig germ line follows the overall dynamics shown in mice, suggesting that epigenetic reprogramming of germ cells is conserved in mammals. A better understanding of the sequential reprogramming of PGC in the pig will facilitate the derivation of embryonic germ cells in this species.

The mammalian immune system eliminates pathogens by generating a specific antibody response. Polyclonality is a key feature of this immune response: the immune system produces antibodies which bind to different structures on a given pathogen thereby increasing the likelihood of its elimination. The vast majority of current recombinant antibody drugs rely on monospecific monoclonal antibodies. Inherently, such antibodies do not represent the benefits of polyclonality utilized by a natural immune system and this has impeded the identification of efficacious antibody drugs against infectious agents, including viruses. The development of novel technologies has allowed the identification and manufacturing of antigen-specific recombinant polyclonal human antibodies, so-called symphobodies. This review describes the rationale for designing drugs based on symphobodies against pathogenic viruses, including HIV, vaccinia and smallpox virus, and respiratory syncytial virus.

The mammalian germline is generally assumed to undergo extensive epigenetic reprogramming during embryonic development, including a nearly complete erasure of DNA methylation. This assumption does, however, to large degree rely on data from mouse, and despite a well-grounded picture the general nature of these data needs to be validated by investigations of other mammalian species. This study represents such a contribution in the examination of the germline in the domestic pig (Sus scrofa). Semiquantitative immunohistochemistry was used to investigate the level of DNA methylation in the POU5F1-positive primordial germ cells (PGCs) compared with neighboring somatic cells in porcine embryos at Embryonic Day 15 (E15), E17, E20, E21, and E28. We show that, in agreement with the mouse model, a significantly lower level of DNA methylation was observed in the early migrating PGCs. This level was decreasing until a stage coinciding with the entrance of the PGCs to the genital ridge. After this, the methylation level increased. Using whole-mount immunostaining, we determined the spatial arrangement of the porcine PGCs in the period between E15 and E28, allowing some comparison with the migration of the murine germline. The overall conclusion from the obtained data is that the DNA methylation changes in porcine PGCs, as well as the migration of these cells, parallels the picture reported for the mouse.

thorax group (trxG) genes, which generally antagonize PcG genes.These results suggest that wge is involved in epigenetic regulation, but its function is different from typical PcG and trxG.In this study, to know whether wge maintains either silenced or active transcriptional states, we investigated the effects of wge mutation on PcG / trxG PcG/trxG response element (PRE / TRE)mediated (PRE/TRE)-mediated gene regulations. Fab-7 is a genetically identified PRE / TRE PRE/TRE of the bithorax-complex. The analysis demonstrated that wge functions as trxG and maintains of active transcriptional state on at least one of PRE / TRE, PRE/ TRE, Fab-7. Next, we performed DNA microarray analysis in order to narrow down the target genes of wge. The analysis revealed that 581 genes was up-regulated and 494 genes was down-regulated by overexpression of wge in eye discs. These genes are candidates for targets of wge including the genes regulated epigenetically by wge in the eye to wing transformation. Now, we are trying to do ChIPon-chip analysis using wge-expression cells.The mammalian germline is known from several species to undergo extensive epigenetic reprogramming during embryonic development, including a nearly complete erasure of DNA methylation. In the murine embryo the erasure is initiated during migration of the primordial germ cells (PGCs) towards the forming gonadal tissue and by E12.5, the level is nearly zero. Mapping of the reprogramming event has hitherto been limited to the mouse.In this study the genomewide DNA methylation in the primordial germ line was examined for the domestic pig. The porcine primordial germline is distinguishable by specific OCT4 expression from around 14 days post insemination (dpi) (pre-somite stage), where the expression of this pluripotency-related transcription factor decreases in all other cell types. In the period between 14 and 19 dpi, the PGCs are located in the yolk sac and the early hind gut mesentery. Subsequently, they migrate towards the genital ridges which they enter during a couple of days.We have used semiquanitative semiquantitative immunohistochemistry to investigate the level of methylated DNA in the PGCs compared to neighbouring somatic cells in porcine embryos at 15, 17, 20, 21 and 28 dpi.In agreement with the mouse model, a relatively lower level of DNA methylation is observed in the PGCs at 15 dpi, further decreasing until 21 dpi after which the level again rises. At 28 dpi, the level is comparable to that of PGCs at 17 dpi. Detailed statistic analysis of the data is being performed and hence, final conclusions will be presented on the poster. Determination, or commitment, of cell fate is a crucial milestone for embryonic cells. We previously showed that, in the mouse limb bud, determination of positional identity occurs as cells gradually lose their intrinsic ability to respond to instructive positional information. The mechanism underlying this loss is scarcely understood, but may be epigenetic in nature. We find that the histone methyltransferase Enhancer of zest...