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SCNT technology has been successfully used to clone a variety of mammals, but the cloning efficiency is very low. This low efficiency is likely due to the incomplete reprogramming of SCNT embryos. Histone modification and DNA methylation may participate in these events. Thus, it would be interesting to attempt to improve the efficiency of SCNT by using a HDACi VPA. In order to guarantee the effect of VPA and reduce its cytotoxicity, a comprehensive analysis of the cell proliferation and histone modification was performed. The results showed that 0.5 and 1 mM VPA treatment for 24 h were the optimal condition. According to the results, H3K4me3 was increased in 0.5 and 1 mM VPA groups, whereas H3K9me2 was significantly decreased. These are the signals of gene-activation. In addition, VPA treatment led to the overexpression of Oct4 and Nanog. These indicated that VPA-treated cells had similar patterns of histone to zygotic embryos, and may be more favorable for reprograming. A total of 833 cloned embryos were produced from the experimental replicates of VPA-treated donor cells. In 1 mM treatment group, the blastocyst rates were significantly increased compared with control. At the same time, our findings demonstrated the interrelation between DNA methylation and histone modifications. [ABSTRACT FROM AUTHOR]

DNA methylation in mammals is an epigenetic marker and necessary for normal embryogenesis. The global genomic demethylation of 5-methylcytosine occurs during the first cell cycle following fertilization. Activation-induced cytidine deaminase (AID), which is well-known for the function in antibody diversification, has been implicated to play a role in active demethylation, but its role in cell reprogramming and its crosstalk with other DNA demethylation mechanism need to be clarified. In this study, the dynamic epigenetic regulation of cell pluripotency and embryo development by AID in bovine preimplantation embryos was investigated. The analysis of an AID overexpressing transgenic cell line showed that AID overexpression did not change the global genomic methylation but did change the methylation status of the promoters of the OCT4, NANOG and SOX2 genes, thereby causing changes in their expression. The siRNA-mediated AID knockdown in early embryonic development indicated that AID interference did not affect oocyte maturation or the following embryo development after in vitro fertilization but influenced the DNA methylation status of OCT4 and NANOG. To clarify the role of AID in preimplantation embryos, SCNT embryos were obtained using AID-overexpressing cells as nuclear donors. Compared to the control group, the cleavage and blastocyst rates were both significantly improved in the AID-overexpression group. The expression of OCT4 and NANOG was increased in the SCNT embryos, whereas the methylation levels of their promoters were reduced. In conclusion, this study demonstrated that AID selectively catalyzes DNA demethylation of pluripotency genes to play a role in regulation their expression, improves bovine SCNT embryo development by increased expression levels. [ABSTRACT FROM AUTHOR]

Our objective is to investigate the effect of valproic acid (VPA), a histone deacetylase inhibitor, on early embryonic development. We studied the effect of VPA on the in vitro maturation of bovine oocytes, and on the development of bovine embryos derived from in vitro fertilization (IVF) or parthenogenesis. Germinal vesicle stage bovine oocytes were cultured with different concentrations of VPA for 24 h; low dose VPA treatment (0.03 and 0.3 mM) had no effect on oocyte maturation, but 3 and 6 mM VPA significantly decreased maturation rate; when used for IVF or parthenogenesis, VPA-treated oocytes generated significantly lowered blastocyst rate. Oocytes matured in vitro were fertilized or underwent parthenogenetic activation; 6 h later, they were exposed to VPA for 48 h, and then the cleavage rate, blastocyst rate and mRNA expression levels of transcription factors ( Oct4, Nanog, and Cdx2) were assessed. For embryos cultured in 0.3 mM VPA, there was no remarkable change in cleavage rate or blastocyst rate, but the expression of Oct4 and Nanog in blastocysts was significantly increased. For embryos treated with 3.0 mM VPA, the cleavage rate and blastocyst rate were significantly decreased. In conclusion, low dose VPA has no effect on oocyte maturation but affects subsequent embryonic development. Low dose VPA administration to IVF embryos had no effect on embryonic development, but the expression of several important transcription factors was increased. Treatment of IVF embryos with low dose VPA may improve their development potential. [ABSTRACT FROM AUTHOR]

In the present study, cashmere goat fetal fibroblasts were transfected with pCDsR-KI, a hair-follicle-cell specific expression vector for insulin-like growth factor 1 (IGF1) that contains two markers for selection (red fluorescent protein gene and neomycin resistant gene). The transgenic fibroblasts cell lines were obtained after G418 selection. Prior to the somatic cell nuclear transfer (SCNT), the maturation rate of caprine cumulus oocytes complexes (COCs) was optimized to an in vitro maturation time of 18 h. Parthenogenetic ooctyes were used as a model to investigate the effect of two activation methods, one with calcium ionophore IA23187 plus 6-DMAP and the other with ethanol plus 6-DMAP. The cleavage rates after 48 h were respectively 88.7% and 86.4%, with no significant difference ( P>0.05). There was no significant difference between the cleavage rate and the blastocyst rate in two different media (SO-Faa and CR1aa; 86.3% vs 83.9%, P>0.05 and 23.1% vs 17.2%, P>0.05). The fusion rate of a 190 V/mm group (62.4%) was significantly higher than 130 V/mm (32.8%) and 200 V/mm (42.9%), groups ( P>0.05). After transgenic somatic cell nuclear transfer (TSCNT) manipulation, 203 reconstructed embryos were obtained in which the cleavage rate after in vitro development (IVD) for 48 h was 79.3% (161/203). The blastocyst rate after IVD for 7 to 9 d was 15.3% (31/203). There were 17 embryos out of 31 strongly expressing red fluorescence. Two of the red fluorescent blastocysts were randomly selected to identify transgene by polymerase chain reaction. Both were positive. These results showed that: (i) RFP and Neo r genes were correctly expressed indicating that transgenic somatic cell lines and positive transgenic embryos were obtained; (ii) one more selection at the blastocyst stage was necessary although the donor cells were transgenic positive, because only partially transgenic embryos expressing red fluorescence were obtained; and (iii) through TSCNT manipulation and optimization, transgenic cashmere goat embryos expressing red fluorescence and containing an IGF1 expression cassette were obtained, which was sufficient for production of transgenic cashmere goats. [ABSTRACT FROM AUTHOR]