Unfaithful Maintenance of Methylation Imprints Due to Loss of Maternal Nuclear Dnmt1 during Somatic Cell Nuclear Transfer

The low success rate of somatic cell nuclear transfer (SCNT) in mammalian cloning is largely due to imprinting problems. However, little is known about the mechanisms of reprogramming imprinted genes during SCNT. Parental origin-specific DNA methylation regulates the monoallelic expression of imprinted genes. In natural fertilization, methylation imprints are established in the parental germline and maintained throughout embryonic development. However, it is unclear whether methylation imprints are protected from global changes of DNA methylation in cloned preimplantation embryos. Here, we demonstrate that cloned porcine preimplantation embryos exhibit demethylation at differentially methylated regions (DMRs) of imprinted genes; in particular, demethylation occurs during the first two cell cycles. By RNAi-mediated knockdown, we found that Dnmt1 is required for the maintenance of methylation imprints in porcine preimplantation embryos. However, no clear signals were detected in the nuclei of oocytes and preimplantation embryos by immunofluorescence. Thus, Dnmt1 is present at very low levels in the nuclei of porcine oocytes and preimplantation embryos and maintains methylation imprints. We further showed that methylation imprints were rescued in nonenucleated metaphase II (MII) oocytes. Our results indicate that loss of Dnmt1 in the maternal nucleus during SCNT significantly contributes to the unfaithful maintenance of methylation imprints in cloned embryos

Tissue engineering, stem cells, cloning, and parthenogenesis: new paradigms for therapy

Patients suffering from diseased and injured organs may be treated with transplanted organs. However, there is a severe shortage of donor organs which is worsening yearly due to the aging population. Scientists in the field of tissue engineering apply the principles of cell transplantation, materials science, and bioengineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. Both therapeutic cloning (nucleus from a donor cell is transferred into an enucleated oocyte), and parthenogenesis (oocyte is activated and stimulated to divide), permit extraction of pluripotent embryonic stem cells, and offer a potentially limitless source of cells for tissue engineering applications. The stem cell field is also advancing rapidly, opening new options for therapy. The present article reviews recent progress in tissue engineering and describes applications of these new technologies that may offer novel therapies for patients with end-stage organ failure

Aberrant epigenetic changes and gene expression in cloned cattle dying around birth

<p>Abstract</p> <p>Background</p> <p>Aberrant reprogramming of donor somatic cell nuclei may result in many severe problems in animal cloning. To assess the extent of abnormal epigenetic modifications and gene expression in clones, we simultaneously examined DNA methylation, histone H4 acetylation and expression of six genes (<it>β-actin</it>, <it>VEGF</it>, <it>oct4</it>, <it>TERT</it>, <it>H19 </it>and <it>Igf2</it>) and a repetitive sequence (<it>art2</it>) in five organs (heart, liver, spleen, lung and kidney) from two cloned cattle groups that had died at different stages. In the ED group (early death, n = 3), the cloned cattle died in the perinatal period. The cattle in the LD group (late death, n = 3) died after the perinatal period. Normally reproduced cattle served as a control group (n = 3).</p> <p>Results</p> <p>Aberrant DNA methylation, histone H4 acetylation and gene expression were observed in both cloned groups. The ED group showed relatively fewer severe DNA methylation abnormalities (p < 0.05) but more abnormal histone H4 acetylations (p < 0.05) and more abnormal expression (p < 0.05) of the selected genes compared to the LD group. However, our data also suggest no widespread gene expression abnormalities in the organs of the dead clones.</p> <p>Conclusion</p> <p>Deaths of clones may be ascribed to abnormal expression of a very limited number of genes.</p

Cytokine Gene Expression in the Maternal-Fetal Interface in Somatic Cell Nuclear Transfer Pregnancies in Small Ruminants

The present retrospective study investigates pregnancy rates, incidence of pregnancy losses and large offspring syndrome (LOS), and immune-related gene expression of sheep and goat somatic cell nuclear transfer (SCNT) pregnancies. We hypothesized that significantly higher pregnancy losses observed in sheep SCNT pregnancies compared to goats are due to the increased amounts of T-helper 1 cytokines and pro-inflammatory mediators at the maternal-fetal interface. Sheep and goat SCNT pregnancies were generated using the same procedure. Control pregnancies were established by natural breeding. Although SCNT pregnancy rates at 45 days were similar in both species, pregnancy losses between 45 and 60 days and incidence of LOS were significantly increased in sheep compared with goats. At term, the expression of pro-inflammatory genes in sheep SCNT placentas was increased while the one of goat SCNT was similar to the control animals. Among the genes that had altered expression in sheep SCNT placentas are CTLA4, IL2RA, CD28, IFNG, IL6, IL10, TGFB1, TNF, IL1A and CXCL8. MHC-I protein expression was greater in sheep and goat SCNT placentas at term compared with control pregnancies. An unfavorable immune environment is present at the maternal-fetal interface in sheep SCNT pregnancies