胚胎发育过程伴随着遗传学与表观遗传学不断变化,表观重编程完整性直接影响胚胎发育能力.孤雌胚胎发育可能与表观重编程不完全密切相关.然而,当前关于兔早期胚胎发育的表观遗传研究鲜有报道.研究拟确定兔早期胚胎中DNA甲基化与组蛋白修饰动态分布,比较这些表观修饰在体内受精胚胎与孤雌胚胎的差异.结果表明:体内受精胚胎与孤雌胚胎均呈现较高H3K4me2和泛乙酰化丰度,而较低H3K27me2和巴豆酰化丰度.四细胞到囊胚时期,孤雌胚胎5-甲基胞嘧啶(5mC)含量高于体内受精胚胎.二细胞到桑椹胚时期,孤雌胚胎5-羟甲基胞嘧啶(5hmC)水平高于体内受精胚胎.培养基添加维生素C并没有显著改善孤雌胚胎DNA甲基化状态,而应用5-Aza-dC处理58 h能够降低5mC和5hmC水平,说明使用化合物进行适当处理可以改善孤雌胚胎的表观修饰水平.

Porcine chimeras are valuable in the study of pluripotency, embryogenesis and development. It would be meaningful to generate chimeric piglets from somatic cell nuclear transfer embryos. In this study, two cell lines expressing the fluorescent markers enhanced green fluorescent protein (EGFP) and tdTomato were used as donor cells to produce reconstructed embryos. Chimeric embryos were generated by aggregating two EGFP-cell derived embryos with two tdTomato-cell derived embryos at the 4-cell stage, and embryo transfer was performed when the aggregated embryos developed into blastocysts. Live porcine chimeras were successfully born and chimerism was observed by their skin color, gene integration, microsatellite loci composition and fluorescent protein expression. The chimeric piglets were largely composed of EGFP-expressing cells, and this phenomenon was possibly due to the hyper-methylation of the promoter of the tdTomato gene. In addition, the expression levels of tumorigenicity-related genes were altered after tdTomato transfection in bladder cancer cells. The results show that chimeric pigs can be produced by aggregating cloned embryos and that the developmental capability of the cloned embryo in the subsequent chimeric development could be affected by the growth characteristics of its donor cell.

The Cre–loxP system is a powerful tool for genetic analysis of distinct cell lineages and tissue‐specific gene knockout in animal models. VASA is specifically expressed in reproductive tissues, and is known to play important roles in spermatogenesis and germ‐cell growth. In this study, Cre recombinase transgenic pigs under the control of the VASA promoter were generated by somatic cell nuclear transfer. Germ cell‐specific expression of Cre recombinase in VASA‐Cre transgenic pigs was shown by western blotting and immunohistochemistry. VASA‐Cre transgenic pigs will be a useful tool for germ cell‐specific gene knockout and a disease model for disorders of the reproductive system.

To understand if the genomic imprinting status of the donor cells is altered during the process of SCNT (somatic cell nuclear transfer), cloned pigs were produced by SCNT using PEF (porcine embryonic fibroblast) and P‐PEF (parthenogenetic‐PEF) cells as donors. Then, the gene expression and methylation patterns of H19, IGF2, NNAT and MEST were compared between PEF vs. C‐PEF (cloned‐PEF), P‐PEF vs. CP‐PEF (cloned‐P‐PEF), respectively. Taken together, the results revealed that there was no significant difference in the expression of imprinted genes and conserved genomic imprints between the donor and cloned cells.

Although the expression and epigenetic status of imprinted genes have been extensively studied in a number of species, less is known about the genomic imprinting in rabbits. Neuronatin (Nnat) plays significant roles in the brain development and metabolic regulation and has been identified to be imprinted and paternally expressed in humans, mice and pigs; however, it has not yet been investigated in rabbits. In this study, we confirmed the expression of two isoforms of the rabbit Nnat (Nnat-a and Nnat-β) identified in Genbank and Ensembl by quantitative real-time PCR. In addition, we also determined the methylation profile of the CpG island in the promoter region of the rabbit Nnat using bisulfite sequencing PCR and combined bisulfite restriction analysis. Here, we provide the first evidence that Nnat has two transcripts in rabbit. Additionally, the CpG island located in the promoter region shows oocyte-specific methylation and may be the differentially methylated region of Nnat in rabbits.

Background/Aims: Parthenogenetic embryos are useful in many applications, such as being an alternative source of embryonic stem cells that would avoid ethical problems. Aberrance in epigenetic reprogramming is considered the major reason for the developmental failure of parthenogenetic embryos. Many histone deacetylase inhibitors have been shown to improve the reprogramming of stem cells and embryos. Here, the relationship between histone modification and parthenogenetic embryonic development was explored. Methods: Valproic acid (VPA) treatment was applied during the culture of parthenogenetic embryos. The abundance of histone modifiers was examined by immunofluorescence and quantified by Image-pro software. Results: The acH3K9 level in in vitro fertilized embryos was significantly higher than parthenogenetic embryos. VPA treatment improved both the blastocyst formation rate and the acH3K9 level in parthenogenetic embryos. The signal intensities of acH4K5 and H3K4me2 were also enhanced in VPA treated embryos. The H3K27me2 level was decreased in the VPA treated embryos at the 2-cell stage. However, the enhancement in the acH3K9, acH4K5 and H3K4me2 level, or the decrease in the H3K27me2 level disappeared shortly after VPA withdrawal. Conclusion: Optimizing histone modifications for a short time following activation was sufficient to enhance the in vitro development of parthenogenetic embryos.

Cloned pigs generated by somatic cell nuclear transfer (SCNT) show a greater ratio of early abortion during mid-gestation than normal controls. X-linked genes have been demonstrated to be important for the development of cloned embryos. To determine the relationship between the expression of X-linked genes and abortion of cloned porcine fetuses, the expression of X-linked genes were investigated by quantitative real-time polymerase chain reaction (q-PCR) and the methylation status of Xist DMR was performed by bisulfate-specific PCR (BSP). q-PCR analysis indicated that there was aberrant expression of X-linked genes, especially the upregulated expression of Xist in both female and male aborted fetuses compared to control fetuses. Results of BSP suggested that hypomethylation of Xist occurred in aborted fetuses, whether male or female. These results suggest that the abnormal expression of Xist may be associated with the abortion of fetuses derived from somatic cell nuclear transfer embryos.

It is generally believed that aberrant expression of imprinted genes participates in growth retardation of mammalian parthenogenesis. Neuronatin (NNAT), a paternally expressed gene, plays important roles in neuronal growth and metabolic regulation. Here we have compared the gene expression and promoter methylation pattern of NNAT between pig normally fertilized (Con) and parthenogenetic (PA) embryos. The results showed loss of NNAT expression (p<0.001) and hypermethylation of NNAT promoter in PA samples. Additionally, partial methylation was observed in Con fetuses, while almost full methylation and unmethylation of NNAT promoter were apparent in Metaphase II (MII) oocytes and mature sperms, respectively, which identified the CpG promoter region as a putative differentially methylated region (DMR) of NNAT. The data demonstrate that promoter hypermethylation is associated with the silencing of NNAT in pig PA fetuses, which may be related to developmental failure of pig parthenogenesis at early stages.

In the present study,to promote the developmental competency of porcine recloned embryos through vaproic acid(VPA) treatment,the treated time and concentration for VPA was firstly exploited.The results showed that the highest blastocyst formation rate could be harvested when treated with 1 mmol/L VPA for 16 h.Through immunostaining,it showed that the fluorescent intensity of acH3K9 was higher in the VPA treated group at blastocyst stage,compared with control.There were no significantly differences between the treated and non-treated group in the gene expression level of Oct4,Nanog,Sox2 and Klf4,as was revealed by Real-time PCR analysis.These results provide evidences that the in vitro developmental competency of porcine recloned embryos could be improved with VPA treatment.

In order to construct cell lines without CD163gene,agene of porcine reproductive and respiratory syndrome virus receptor,and find out role of CD163in PRRSV infection,5'and 3'homologous arms of CD163gene of porcine reproductive and respiratory syndrome virus receptor were amplified and cloned into pGEM-T vector.Results of sequencing showed that 5'and 3'homologous arms of CD163gene were 4.5kb and 1.1kb,respectively.Then,the homologous arms of CD163gene were subcloned into pSSC-9to construct a targeting vector pSSC-Larm-Sarm for CD163gene knock-out.The vector will be used to construct fibroblast cells with CD163gene knockout.