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Xu W.,South China Agricultural University | Xu W.,Longyan University | Li Z.,South China Agricultural University | Yu B.,South China Agricultural University | And 6 more authors.
PLoS ONE | Year: 2013

Somatic cell nuclear transfer (SCNT) in mammalian cloning currently remains inefficient. Incomplete or erroneous epigenetic reprogramming of specialized donor somatic nuclear and resulting aberrant gene expression during development of cloned embryos is commonly believed as the main reason that causes the low efficiency of SCNT. Use of small molecular reprogramming modifiers to assist the somatic nucleus to mimic naturally occurring DNA methylation and chromatin remodeling in nucleus of fertilization-derived zygotes, has been widely attempted to improve cloning efficiency. However, impacts of these small modifiers on gene-specific methylation dynamics and their potential effects on methylation of imprinted gene have rarely been traced. Here, we attempted two relatively novel DNMT1 inhibitor (DNMTi) and histone deacetylase inhibitor (HDACi), scriptaid and RG108, and demonstrated their effects on dynamics of gene-specific DNA methylation and transcription of porcine SCNT embryos. We found that scriptaid and RG108 had synergetic effects on rescuing the disrupted methylation imprint of H19 during SCNT at least partially by repression over-expressed MBD3 in eight-cell cloned embryos. Furthermore, we firstly identified a differential methylation regions (DMRs) at 5′ flanking regions of XIST gene and found that scriptaid alone and its combination with RG108 modify the dynamics of both transcription and DNA methylation levels in cloned embryos, by different manners. Additionally, we found that scriptaid alone and its combination with RG108 can significantly promote the transcription of NANOG in cloned embryos and enhance their pre-implantation developmental capacity. Our results would contribute to uncovering the epigenetic reprogramming mechanisms underlying the effects of assisted small molecules on improvement of mammalian cloning efficiency. © 2013 Xu et al.

Xu W.-H.,South China Agricultural University | Xu W.-H.,Longyan University | Li Z.-C.,South China Agricultural University | Ouyang Z.-P.,South China Agricultural University | And 5 more authors.
Journal of Integrative Agriculture | Year: 2015

Somatic nuclear transfer technology has become increasingly promising in biomedicine and agriculture. Whereas the approach remains inefficient and underlying mechanisms remain ambiguous. Although cloned embryos have similar in vitro developmental capacity as in vitro fertilized (IVF) embryos before implantation, they appeared to have much lower full-term developmental efficiency in pig and cattle, and thus it would be reasonable to postulate that profound distinction at the molecular level should exist between them. Herein, RNA sequencing technique was used to screen differentially expressed genes in cloned and IVF blastocysts, and in total 628 differentially expressed transcripts were obtained, among which, 280 transcripts are up-regulated and 348 transcripts are down-regulated in cloned blastocysts. Moreover, one statistically significant pathway associated with endoplasmic reticulum (ER) protein processing was enriched, and some ER-stress markers such as ATF4, ATF6, PDIA3, HSPA1B, HSP40 and HSP90 between cloned and IVF blastocysts were suggested. Additionally, some developmentally important genes such as lipid metabolism related genes (MGLL, DDHD2 and FADS2) and epigenetic modification genes (DNMT1, KDM5C and MBD3L5) were found differentially expressed between cloned and IVF embryos. © 2015 Chinese Academy of Agricultural Sciences.

Li Z.,South China Agricultural University | He X.,South China Agricultural University | He X.,Wens Research Institute | Chen L.,South China Agricultural University | And 5 more authors.
Cellular Reprogramming | Year: 2013

The somatic cell nuclear transfer (SCNT) technique has been widely applied to clone pigs or to produce genetically modified pigs. Currently, this technique relies mainly on using terminally differentiated fibroblasts as donor cells. To improve cloning efficiency, only partially differentiated multipotent mesenchymal stem cells (MSCs), thought to be more easily reprogrammed to a pluripotent state, have been used as nuclear donors in pig SCNT. Although in vitro-cultured embryos cloned from porcine MSCs (MSCs-embryos) were shown to have higher preimplantation developmental ability than cloned embryos reconstructed from fibroblasts (Fs-embryos), the difference in in vivo full-term developmental rate between porcine MSCs-embryos and Fs-embryos has not been investigated so far. In this study, we demonstrated that blastocyst total cell number and full-term survival abilities of MSCs-embryos were significantly higher than those of Fs-embryos cloned from the same donor pig. The enhanced developmental potential of MSCs-embryos may be associated with their nuclear donors' DNA methylation profile, because we found that the methylation level of imprinting genes and repeat sequences differed between MSCs and fibroblasts. In addition, we showed that use of transgenic porcine MSCs generated from transgene plasmid transfection as donor cells for SCNT can produce live transgenic cloned pigs. These results strongly suggest that porcine bone marrow MSCs are a desirable donor cell type for production of cloned pigs and genetically modified cloned pigs via SCNT. © Mary Ann Liebert, Inc.

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