Argonaute 2 is a key regulator of maternal mRNA degradation in mouse early embryos.

In mammalian early embryos, the transition from maternal to embryonic control of gene expression requires timely degradation of a subset of maternal mRNAs (MRD). Recently, zygotic genome activation (ZGA)-dependent MRD has been characterized in mouse 2-cell embryo. However, in early embryos, the dynamics of MRD is still poorly understood, and the maternal factor-mediated MRD before and along with ZGA has not been investigated.

Endo-siRNAs repress expression of SINE1B during in vitro maturation of porcine oocyte.

Approximately 40% of mammalian genome is made of transposable elements (TEs), and during specific biological processes, such as gametogenesis, they may be activated by global demethylation, so strict silencing mechanism is indispensable for genomic stability. Here, we performed small RNA-seq on Dicer1 knockdown (KD) oocytes in pig, and observed short interspersed nuclear elements 1B (SINE1B) derived endogenous small interfering RNAs (endo-siRNAs), termed SINE1B-siRNAs, were significantly decreased and their biogenesis was dependent on Dicer1 and transcript of SINE1B. Furthermore, by injection of mimics and inhibitors of the SINE1B-siRNAs into germinal vesicle-stage (GV-stage) oocytes, we found the maturation rate was significantly decreased by SINE1B-siRNAs, indicating the SINE1B-siRNAs are indispensible for in vitro maturation (IVM) of porcine oocyte.

Alteration of the DNA methylation status of donor cells impairs the developmental competence of porcine cloned embryos.

Nuclear reprogramming induced by somatic cell nuclear transfer is an inefficient process, and donor cell DNA methylation status is thought to be a major factor affecting cloning efficiency. Here, the role of donor cell DNA methylation status regulated by 5-aza-2'-deoxycytidine (5-aza-dC) or 5-methyl-2'-deoxycytidine-5'-triphosphate (5-methyl-dCTP) in the early development of porcine cloned embryos was investigated. Our results showed that 5-aza-dC or 5-methyl-dCTP significantly reduced or increased the global methylation levels and altered the methylation and expression levels of key genes in donor cells.

Epigenetic modification agents improve genomic methylation reprogramming in porcine cloned embryos.

Incomplete DNA methylation reprogramming in cloned embryos leads to low cloning efficiency. Our previous studies showed that the epigenetic modification agents 5-aza-2'-deoxycytidine (5-aza-dC) or trichostatin A (TSA) could enhance the developmental competence of porcine cloned embryos. Here, we investigated genomic methylation dynamics and specific gene expression levels during early embryonic development in pigs.

Treating cloned embryos, but not donor cells, with 5-aza-2'-deoxycytidine enhances the developmental competence of porcine cloned embryos.

The efficiency of cloning by somatic cell nuclear transfer (SCNT) has remained low. In most cloned embryos, epigenetic reprogramming is incomplete, and usually the genome is hypermethylated. The DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) could improve the developmental competence of cow, pig, cat and human SCNT embryos in previous studies.

[Telomere lengthening by trichostatin A treatment in cloned pigs].

Telomeres are repeated GC rich sequences at the end of chromosomes, and shorten with each cell division due to DNA end replication problem. Previously, reprogrammed somatic cells of cloned animals display variable telomere elongation. However, it was reported that the cloned animals including Dolly do not reset telomeres and show premature aging.

[TSA improve transgenic porcine cloned embryo development and transgene expression].

Uncompleted epigenetic reprogramming is attributed to the low efficiency of producing transgenic cloned animals. Histone modification associated with epigenetics can directly influence the embryo development and transgene expression. Trichostatin A (TSA), as an inhibitor of histone deacetylase, can change the status of histone acetylation, improve somatic cell reprogramming, and enhance cloning efficiency.

Overexpression Nanog activates pluripotent genes in porcine fetal fibroblasts and nuclear transfer embryos.

Nanog as an important transcription factor plays a pivotal role in maintaining pluripotency and in reprogramming the epigenome of somatic cells. Its ability to function on committed somatic cells and embryos has been well defined in mouse and human, but rarely in pig. To better understand Nanog's function on reprogramming in porcine fetal fibroblast (PFF) and nuclear transfer (NT) embryo, we cloned porcine Nanog CDS and constructed pcDNA3.

[Inheritance and expression stability of transgene in transgenic animals].

Transgenic technology is one of the most hotspots in biology. In the past decade, the progress in animal cloning has provided an alternative method to improve transgenic efficiency. Many kinds of transgenic animals have been successfully produced via the combination of transfection and nuclear transfer.

Effect of chilling on porcine germinal vesicle stage oocytes at the subcellular level.

The potential subcellular consequence of chilling on porcine germinal vesicle (GV) stage oocytes was examined. Prior to in vitro maturation (IVM), Cumulus-oocyte complexes (COCs) freshly collected from antral follicles (3-6mm in diameter) were evenly divided into four groups and immediately incubated in PVA-TL-HEPES medium at the temperature of 39 degrees C (control group), 23 degrees C (room temperature), 15 degrees C and 10 degrees C for 10min, respectively. Following 42h of IVM at 39 degrees C, the survival rates were examined.

[Culture of porcine mesenchymal stem cells in vitro and developmental capacity of reconstructed embryos by nuclear transfer].

The developmental potential of reconstructed embryos varied according to the source of donor cells, it was thought that the donor cells capabilities to be reprogrammed were different. We established the method of culturing porcine bone marrow mesenchymal stem cells (pMSCs), identified and observed the growth characteristics of pMSCs, and determined pMSCs reprogramming potential as donor cells for nuclear transfer (SCNT). We found that the method of gradient centrifugation to isolate pMSCs from porcine bone marrow was better than the method of anchoring culture; the number of pMSCs achieved peak at day 6, the adhesive rate of cultured cells was 78.