Dean, W. et al. Conservation of methylation reprogramming in mammalian development: aberrant reprogramming in cloned embryos. Proc. Natl Acad. Sci. USA 98, 13734-13738

Laboratory of Developmental Genetics and Imprinting, Developmental Genetics Program, Babraham Institute, Cambridge CB2 4AT, United Kingdom.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 12/2001; 98(24):13734-8. DOI: 10.1073/pnas.241522698
Source: PubMed

ABSTRACT Mouse embryos undergo genome-wide methylation reprogramming by demethylation in early preimplantation development, followed by remethylation thereafter. Here we show that genome-wide reprogramming is conserved in several mammalian species and ask whether it also occurs in embryos cloned with the use of highly methylated somatic donor nuclei. Normal bovine, rat, and pig zygotes showed a demethylated paternal genome, suggesting active demethylation. In bovine embryos methylation was further reduced during cleavage up to the eight-cell stage, and this reduction in methylation was followed by de novo methylation by the 16-cell stage. In cloned one-cell embryos there was a reduction in methylation consistent with active demethylation, but no further demethylation occurred subsequently. Instead, de novo methylation and nuclear reorganization of methylation patterns resembling those of differentiated cells occurred precociously in many cloned embryos. Cloned, but not normal, morulae had highly methylated nuclei in all blastomeres that resembled those of the fibroblast donor cells. Our study shows that epigenetic reprogramming occurs aberrantly in most cloned embryos; incomplete reprogramming may contribute to the low efficiency of cloning.

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Available from: Jörn E Walter, Jul 29, 2015
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    • "The morphology of cloned embryos and their gene expression are often abnormal [12]. One means to improve embryo quality is aggregating two or three cleavage-stage cloned embryos, which increases the embryo size and pregnancy rate [14,15,20–22]. "
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    ABSTRACT: The production of cloned embryos using conventional methods has extremely low success rates owing to low embryo quality. To improve the quality of cloned bovine embryos expressing enhanced green fluorescent protein (EGFP), we applied an aggregation culture method. The EGFP gene was transfected into bovine fetal fibroblasts using a retroviral vector system. Somatic cell nuclear transfer was performed using these cells, and the resulting embryos were cultured in aggregates or individually. Gene expression was analyzed by a microarray, and differentially expressed genes were validated by quantitative real-time polymerase chain reaction. The total number of cells per blastocyst and the ratio of inner cell mass cells to trophectoderm cells were higher in aggregated transgenic cloned blastocysts (agBL; 368.7 ± 109.6 and 1:4.8, respectively) than in in vitro-fertilized blastocysts (ivfBL; 189.8 ± 65.8 and 1:2.6, respectively) and nonaggregated transgenic cloned blastocysts (sBL; 113.1 ± 36.3 and 1:1.5, respectively; P < 0.05 and P < 0.01, respectively). Moreover, the blastocyst perimeter was larger in the agBL group than in the ivfBL and sBL groups (1168.8 ± 200.23 vs. 887.33 ± 187.62 and 678 ± 226.1 μm; P < 0.05). In addition, mitochondrial fluorescence intensity was higher in the agBL group than in the ivfBL and sBL groups (P < 0.05). The number of apoptotic cells per blastocyst was lower in the ivfBL and agBL groups than in the sBL group (3.7 ± 2.2 and 3.4 ± 2.1 vs. 6.7 ± 6.8; P < 0.05). The genes identified in the microarray belonged to 18 categories. Expression of the Krüppel-like factor 4 gene, which is associated with cell proliferation, development, and transcription, was 7.2-fold higher in the agBL group than in the ivfBL group (P < 0.05) but did not differ between the sBL and ivfBL groups (P > 0.05). Expression of the heat shock 70-kDa protein 1A gene, which is associated with apoptosis, was 12-fold higher in the sBL group than in the ivfBL and agBL groups (P < 0.05). Expression of a stemness-related gene (octamer-binding transcription factor 4) and trophectoderm-specific genes (homeobox protein CDX2 and keratin 18) was higher in the agBL group than in the sBL group (P < 0.05). However, expression of the stemness gene homeobox protein NANOG did not differ among the groups (P > 0.05). Taken together, these data suggest that the aggregation method improves the quality of cloned embryos expressing EGFP and might be helpful in animal cloning. Copyright © 2015 Elsevier Inc. All rights reserved.
    Theriogenology 04/2015; DOI:10.1016/j.theriogenology.2015.04.008 · 1.85 Impact Factor
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    • "al growing oocyte ( Hiura et al . , 2006 ; O &apos; Doherty et al . , 2012 ) . Following fertilization there is a global cascade of DNA demethylation during the early stages of embryogenesis , whereby the paternal genome is rapidly demethylated in the zygote and the maternal genome is passively demethy - lated in a replication - dependent manner ( Dean et al . , 2001 ; Yang et al . , 2007 ; Iqbal et al . , 2011 ) . More recently , it has been hypothesized that both the maternal and paternal genomes undergo global active demethylation and replication - mediated passive demethylation ( Gkountela and Clark , 2014 ; Guo et al . , 2014 ) . Irrespective of the mechanisms con - trolling these genome - wide"
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    ABSTRACT: Monoallelically expressed genes that exert their phenotypic effect in a parent-of-origin specific manner are considered to be subject to genomic imprinting, the most well understood form of epigenetic regulation of gene expression in mammals. The observed differences in allele specific gene expression for imprinted genes are not attributable to differences in DNA sequence information, but to specific chemical modifications of DNA and chromatin proteins. Since the discovery of genomic imprinting some three decades ago, over one hundred imprinted mammalian genes have been identified and considerable advances have been made in uncovering the molecular mechanisms regulating imprinted gene expression. While most genomic imprinting studies have focused on mouse models and human biomedical disorders, recent work has highlighted the contributions of imprinted genes to complex trait variation in domestic livestock species. Consequently, greater understanding of genomic imprinting and its effect on agriculturally important traits is predicted to have major implications for the future of animal breeding and husbandry. In this review, we discuss genomic imprinting in mammals with particular emphasis on domestic livestock species and consider how this information can be used in animal breeding research and genetic improvement programs.
    Frontiers in Genetics 04/2015; 6. DOI:10.3389/fgene.2015.00156/abstract
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    • "This points to a more moderate rate or a delay in demethylation induced by oxidative stress. A similar trend was found in cloned bovine embryos (Bourc&apos;his et al. 2001; Dean et al. 2001). Conversely, higher "
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    ABSTRACT: Retrotransposons are transposable elements that insert extra copies of themselves throughout the genome via an RNA intermediate using a 'copy and paste' mechanism. They account for more than 44% of the bovine genome and have been reported to be functional, especially during preimplantation embryo development. In the present study, we tested whether high oxygen tension (20% O2) influences global DNA methylation analysed by immunofluorescence staining of developing bovine embryos and whether this has an effect on the expression of some selected retrotransposon families. High oxygen tension significantly increased global DNA methylation in 4-cell embryos and blastocysts. A significant expression difference was observed for ERV1-1-I_BT in female blastocysts, but no significant changes were observed for the other retrotransposon families tested. Therefore, the study indicates that global DNA methylation is not necessarily correlated with retrotransposon expression in bovine preimplantation embryos.
    Reproduction Fertility and Development 12/2014; DOI:10.1071/RD14133 · 2.58 Impact Factor
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