Epigenetic alteration of the donor cells does not recapitulate the reprogramming of DNA methylation in cloned embryos

Abstract. We examined the comprehensive epigenetic status, including histone H3 and H4 acetylation, DNA methylation and level of mRNA transcripts of bovine somatic cell nuclear transfer (SCNT) embryos treated with trichostatin A (TSA), along with their full-term developmental efficacy. Treatment with 50 nM TSA enhanced early developmental competence; increased acetylation of two histones, H3K9K14 and H4K8, at the blastocyst stage; and maintained the DNA methylation status of the satellite I sequence in bovine SCNT embryos. The difference in IGFBP-3 transcript levels between in vivo and SCNT embryos disappeared in SCNT embryos after treatment with 50 nM TSA. Pregnancy, full-term developmental competence and body weight at birth of offspring did not differ between SCNT embryos treated with 50 nM TSA and untreated embryos. These results suggest that treatment with TSA improves preimplantation development and changes the epigenetic status but does not promote the full-term development competence in bovine SCNT embryos. Key words: Bovine embryo, Gene expression, Histone modification, Nuclear transfer, TSA (J. Reprod. Dev. 58: [302][303][304][305][306][307][308][309] 2012) T he efficiency of animal production by somatic cell nuclear transfer (SCNT) is still very low. In bovine cloning, high rates of embryonic, fetal, neonatal and postnatal abnormalities have consistently been observed [1][2][3][4][5][6]. Although the cause of such abnormalities is unknown, the phenomenon may be correlated with abnormal gene expression [7], because several investigators have reported aberant transcription patterns of various genes that have an important role in pre-or postimplantation development in bovine SCNT embryos [8][9][10][11][12][13]. Moreover, an aberrant pattern of gene expression is persistently observed in bovine SCNT embryos that develop to the elongated stage and into a conceptus after implantation [11,12,14,15].The characteristics of gene transcription in SCNT embryos can be attributed to abnormalities in the control system for gene expression such as DNA methylation [16][17][18][19] and acetylation of histones [20]. Kang et al. [16,18] reported that various repeated genomic sequences, including satellite I, were barely demethylated in bovine SCNT embryos at the blastocyst stage. The methylation patterns observed in such embryos closely resembled those in donor cells but were quite different from those in normal embryos produced in vivo or in vitro. These findings suggest dysregulation of epigenetic modifications, such as DNA methylation, resulting in abnormal transcription of developmentally crucial genes at the blastocyst stage.Recently, it was clearly demonstrated in mice that histone modifications are closely related to the success rate of cloning [21,22]. It was reported that treatment with trichostatin A (TSA), which is a histone deacetylase inhibitor, improves in vitro blastocyst development, embryonic stem cell derivation and full-term development of mouse SCNT embryos. Furthermore, TSA treatment also improves...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 95%

Epigenetic Status and Full-term Development of Bovine Cloned Embryos Treated with Trichostatin A

Sawai

Fujii

Hirayama

et al. 2012

“…In serial cloning, accumulation of epigenetic modifications was initially thought to make it impossible to clone past several repetitions [110,111]. However, with recent advances in SCNT, it may be possible to repair epigenetic modifications by chemical treatment [112][113][114][115][116][117][118]. Thus, somatic cell cloning may be repeated beyond previously expected limits (Wakayama, personal communication).…”

Section: Development Of Genetically Modified Pigs For Xenotransplantamentioning

confidence: 99%

Application of Genetically Modified and Cloned Pigs in Translational Research

Matsunari

Nagashima

2009

Abstract. Pigs are increasingly being recognized as good large-animal models for translational research, linking basic science to clinical applications in order to establish novel therapeutics. This article reviews the current status and future prospects of genetically modified and cloned pigs in translational studies. It also highlights pigs specially designed as disease models, for xenotransplantation or to carry cell marker genes. Finally, use of porcine somatic stem and progenitor cells in preclinical studies of cell transplantation therapy is also discussed. Key words: Cloned pig, Gene knockout, Transgenic pig, Translational research (J. Reprod. Dev. 55: [225][226][227][228][229][230] 2009) t present, pigs are not only considered important livestock but are also essential large-animal models in various types of biomedical research [1][2][3][4][5][6]. Furthermore, due to recent technological advances in genetic modification and somatic cell cloning, the range of applications for porcine models has increased markedly [7][8][9][10][11][12][13][14]. The present review focuses on development of genetically modified and cloned pigs and their use in translational studies.Translational research plays an important role as a bridge between basic science outcomes and the clinical realm, leading to development of novel therapeutics. However, disparate findings for rodent models and humans have hindered the translation of rodent data into actionable technologies for patients [15]. In contrast, pigs have many anatomical and physiological similarities to humans, including their cardiovascular systems, omnivorous gastrointestinal tracts and central nervous systems. Their physical sizes are also more comparable to that of humans, rendering pigs more appropriate for development of new surgical or endoscopic techniques.In the future, more advanced translational research may be conducted by improving the clinical relevance of pig models through genetic engineering. This article reviews the current status and future prospects of genetically modified pigs in preclinical studies of stem and progenitor cell therapy, the development of disease models and xenotransplantation.

“…However, this process needed for a differentiated donor nucleus to achieve embryonic status is delayed and incomplete in NT embryos [5]. In fact, many studies have demonstrated that epigenetic statuses such as DNA methylation and histone acetylation, are abnormally established in NT embryos during preimplantation development [6][7][8][9][10][11][12]. As a result, abnormal gene expression including imprinted genes [10,[13][14][15] can prevent normal development of NT embryos [16].…”

mentioning

confidence: 99%

“…Therefore, the level of DNA methylation in normal embryos during preimplantation development is relatively low [22,23]. However, previous studies have shown that the level of DNA methylation in NT embryos is higher than in normal embryos and is more similar to somatic cells, since a somatic genome that is injected into an oocyte does not undergo demethylation [6][7][8][9]11]. This hypermethylation phenomenon is frequently associated with the genome of NT embryos and is considered one of the reasons for abnormal gene expression and low cloning efficiency [21].…”

mentioning

confidence: 99%

Effects of Downregulating DNA Methyltransferase 1 Transcript by RNA Interference on DNA Methylation Status of the Satellite I Region and In Vitro Development of Bovine Somatic Cell Nuclear Transfer Embryos

Yamanaka

Sakatani

Kubota

et al. 2011

Abstract. For the successful production of cloned animals by somatic cell nuclear transfer (NT), the epigenetic status of the differentiated donor cell is reversed to an embryonic totipotent status. However, in NT embryos, this process is aberrant, with genomic hypermethylation consistently observed. Here, we investigated the effects of silencing DNA methyltransferase 1 (DNMT1) mRNA by small interfering RNA (siRNA) on the DNA methylation status of the satellite I region and in vitro development of bovine NT embryos. First, the levels of DNMT1 expression were analyzed at 0, 24, 48, 72, 120 and 192 h after in vitro culture. Real-time PCR and western blotting analyses detected a significant decrease in DNMT1 mRNA in the siRNA-injected NT (siRNA-NT) group up to 72 h after in vitro culture. Next, the levels of DNA methylation of the satellite I region were analyzed at several time points after in vitro culture. The level of DNA methylation detected in siRNA-NT embryos was significantly less than those in NT embryos throughout in vitro development. Moreover, the developmental rate of embryos to blastocysts in the siRNA-NT group was significantly higher than that of NT embryos. Our data suggest that knockdown of DNMT1 mRNA in NT embryos can induce DNA demethylation, which may enhance reprogramming efficiency. Key words: DNA methylation, DNA methyltransferase, Embryo development, Nuclear transfer, RNA interference (J. Reprod. Dev. 57: [393][394][395][396][397][398][399][400][401][402] 2011) hile there have been many attempts to improve the developmental competence of somatic cell nuclear transfer (NT) embryos, the success rate of producing viable offspring is still very low, with less than 5% of embryos transferred to surrogate females [1]. In bovine cloning, high rates of embryonic, fetal, neonatal and postnatal abnormalities have been consistently observed [2][3][4]. To achieve developmental competence of NT embryos, the donor nucleus of the differentiated cell needs to undergo epigenetic reprogramming during preimplantation development. However, this process needed for a differentiated donor nucleus to achieve embryonic status is delayed and incomplete in NT embryos [5]. In fact, many studies have demonstrated that epigenetic statuses such as DNA methylation and histone acetylation, are abnormally established in NT embryos during preimplantation development [6][7][8][9][10][11][12]. As a result, abnormal gene expression including imprinted genes [10,[13][14][15] can prevent normal development of NT embryos [16].DNA methylation of cytosine residues in CpG dinucleotides is one of the epigenetic modifications mediated by three DNA methyltransferases (DNMTs), DNMT1, DNMT3a and DNMT3b [17]. DNA methylation patterns are dynamic, yet tightly regulated, during mammalian development [18]. In normal embryos, both maternal and paternal gametes undergo extensive demethylation after fertilization, with the paternal genome undergoing demethylation within hours of fertilization [19]. In contrast, the maternal genome is passively...