Production of cloned calves by combination treatment of both donor cells and early cloned embryos with 5-aza-2/-deoxycytidine and trichostatin A

Wang, Y.S.; Xiong, Xianrong; An, Zhixing; Wang, L.J.; Liu, J.; Quan, Fusheng; Shi, Hua; Zhang, Yong

doi:10.1016/j.theriogenology.2010.10.022

Cited by 66 publications

(45 citation statements)

References 25 publications

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“…Furthermore, there is also a possibility that long-term treatment of donor cells and reconstructed embryos with TSA is more effective in improving the development of bovine SCNT embryos. However, various abnormalities were also observed after long-term 5-aza-dC and TSA treatment, and large offspring syndrome occurred in nearly all cloned calves [48]. This result suggested that it is difficult to regulate epigenetic status in bovine SCNT embryos by artificial procedures.…”

Section: Discussionmentioning

confidence: 99%

“…It is possible that implantation failure and abortion occurred because of remaining abnormalities in the expression of genes that regulate implantation, placental formation and/or fetal growth. Wang et al [48] reported that combined treatment of both donor cells and reconstructed embryos with 5-aza-2'-deoxycytidine (5-aza-dC) and TSA improved the full-term development of bovine SCNT embryos. Together with the results from our study, both DNA demethylation and histone acetylation may be necessary for normalization of the epigenetic status in bovine SCNT embryos.…”

Section: Discussionmentioning

confidence: 99%

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

Sawai

Fujii

Hirayama

et al. 2012

Journal of Reproduction and Development

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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...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

Sawai

Fujii

Hirayama

et al. 2012

Journal of Reproduction and Development

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“…Moreover, Xist knockdown had a synergistic effect with TSA, resulting in a birth rate of as high as 20% per embryo transferred. This combination would be a promising strategy for largeanimal cloning, where histone deacetylase inhibitors have also proved effective for promoting the development of clones (29,30). Unlike cloning mice, cloning domestic species by SCNT has often been associated with stillbirth or early neonatal death, which is also one of the major obstacles of SCNT that hamper its broad practical application (1).…”

Section: Discussionmentioning

confidence: 99%

RNAi-mediated knockdown of Xist can rescue the impaired postimplantation development of cloned mouse embryos

Matoba

Inoue

Kohda

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

140

163

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Cloning mammals by somatic cell nuclear transfer (SCNT) is highly inefficient. Most SCNT-generated embryos die after implantation because of unidentified, complex epigenetic errors in the process of postimplantation embryonic development. Here we identify the most upstream level of dysfunction leading to impaired development of clones by using RNAi against Xist, a gene responsible for X chromosome inactivation (XCI). A prior injection of Xist-specific siRNA into reconstructed oocytes efficiently corrected SCNT-specific aberrant Xist expression at the morula stage, but failed to do so thereafter at the blastocyst stage. However, we found that shortly after implantation, this aberrant XCI status in cloned embryos had been corrected autonomously in both embryonic and extraembryonic tissues, probably through a newly established XCI control for postimplantation embryos. Embryo transfer experiments revealed that siRNA-treated embryos showed 10 times higher survival than controls as early as embryonic day 5.5 and this high survival persisted until term, resulting in a remarkable improvement in cloning efficiency (12% vs. 1% in controls). Importantly, unlike control clones, these Xist-siRNA clones at birth showed only a limited dysregulation of their gene expression, indicating that correction of Xist expression in preimplantation embryos had a long-term effect on their postnatal normality. Thus, contrary to the general assumption, our results suggest that the fate of cloned embryos is determined almost exclusively before implantation by their XCI status. Furthermore, our strategy provides a promising breakthrough for mammalian SCNT cloning, because RNAi treatment of oocytes is readily applicable to most mammal species.somatic cell cloning | RNA FISH | DNA micro array | Tsix | trichostatin A S omatic cell nuclear transfer (SCNT) is a unique technology for endowing the somatic cell genome with totipotency by genomic reprogramming (1). This technique has a distinct advantage over other similar biotechnologies, such as generating induced pluripotent stem cells, because a single somatic cell can give rise to a new individual with exactly the same genome as that of the donor cell (2, 3). Therefore, SCNT could become an invaluable tool with a broad range of applications including biological drug manufacturing, regenerative medicine, endangered species preservation, and commercial animal breeding. However, the success rate of cloning mammals by SCNT is low, predominantly because of the developmental arrest of cloned embryos after embryo transfer (1, 4).Our recent in-depth gene expression analysis of cloned mouse blastocysts revealed ectopic expression of the noncoding RNA Xist-responsible for X chromosome inactivation (XCI) in female cells (5)-from the active X chromosome in both male and female clones (6). This ectopic expression of Xist had a global adverse effect on the gene expression of cloned embryos, because normalization of Xist expression by genetic knockout remarkably improved the expression patterns of not only X-li...

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“…The procedures of SCNT were performed as described previously (Wang et al 2011). After culturing for 38 h to 42 h, oocytes with expanded cumulus cells were briefly treated with 0.1% (w/v) hyaluronidase and pipetting.…”

Section: Nuclear Transfermentioning

confidence: 99%

Impact of different sources of donor cells upon the nuclear transfer efficiency in Chinese indigenous Meishan pig

Hua¹,

Xu²,

Liu³

et al. 2016

Polish Journal of Veterinary Sciences

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Somatic cell nuclear transfer (SCNT) is currently the most efficient and precise method to generate genetically tailored pig models for both agricultural and biomedical research. However, its efficiency is crucially dependent on the source of nuclear donor cells. In this study, we compared the cloning efficiency by using three lines of donor cells that are derived from fetal, newborn and adult fibroblasts of Chinese indigenous Meishan pig. We showed that cleavage rate and blastocyst formation rate of the reconstructed embryos were not significantly different between the fetal (80.7% and 15.6%) and newborn ear skin (77.5% and 12.3%) fibroblast groups (p>0.05), but in both groups these indices were significantly higher than that found in the adult ear skin (70.5% and 8.8%; p<0.05). Reconstructed embryos derived from fetal, newborn, and adult ear skin fibroblasts were transferred to four surrogates, respectively. For the fetal, newborn, and adult ear skin fibroblasts, the number of pregnancies were two (50.0%), two (50.0%), and one (25.0%), respectively, and the number of deliveries were two (50.0%), one (25.0%), and zero (0.0%), respectively. Seven and two cloned piglets were obtained from the fetal and newborn ear skin fibroblasts respectively, while no piglets were obtained from the adult ear skin fibroblasts. Two cloned piglets from the newborn ear skin fibroblasts died shortly after birth because of neonatal asphyxia caused by dystocia. The birth weights of the piglets derived from the fetal and newborn ear skin fibroblasts were 1230.5 and 1310.0g, respectively, which were statistically insignificant (p>0.05), but both were significantly higher than that of the control groups (p<0.05). Microsatellite analyses demonstrated that the genotypes of all cloned piglets were identical to their donor cells. Therefore, cloned pigs were successfully produced using two sources of donor cells isolated from the fetal and newborn ear skin fibroblasts of Meishan piglet, and indicating a better cloning efficiency than that obtained from adult fibroblasts. We concluded that the nuclear donor cell lines have significant impact on the developmental competence of cloned embryos as well as on the cloning efficiency of Meishan pig.

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Production of cloned calves by combination treatment of both donor cells and early cloned embryos with 5-aza-2/-deoxycytidine and trichostatin A

Cited by 66 publications

References 25 publications

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

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

RNAi-mediated knockdown of Xist can rescue the impaired postimplantation development of cloned mouse embryos

Impact of different sources of donor cells upon the nuclear transfer efficiency in Chinese indigenous Meishan pig

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