Transgenic animals have been used for years to study gene function, produce important proteins, and generate models for the study of human diseases. However, inheritance and expression instability of the transgene in transgenic animals is a major limitation. Copy number and promoter methylation are known to regulate gene expression, but no report has systematically examined their effect on transgene expression. In the study, we generated two transgenic pigs by somatic cell nuclear transfer (SCNT) that express green fluorescent protein (GFP) driven by cytomegalovirus (CMV). Absolute quantitative real-time PCR and bisulfite sequencing were performed to determine transgene copy number and promoter methylation level. The correlation of transgene expression with copy number and promoter methylation was analyzed in individual development, fibroblast cells, various tissues, and offspring of the transgenic pigs. Our results demonstrate that transgene expression is associated with copy number and CMV promoter methylation in transgenic pigs.

The inner cell mass (ICM) in blastocyst is the origin of all somatic and germ cells in mammals and pluripotent stem cells (PSCs) in vitro. As the conserved principles between pig and human, here we performed comprehensive single‐cell RNA‐seq for porcine early embryos from oocyte to early blastocyst (EB). We show the specification of the ICM and trophectoderm in morula and the molecular signature of the precursors. We demonstrate the existence of naïve pluripotency signature in morula and ICM of EB, and the specific pluripotent genes and the activity of signalling pathways highlight the characteristics of the naïve pluripotency. We observe the absence of dosage compensation with respect to X‐chromosome (XC) in morula, and incomplete dosage compensation in the EB. However, the dynamics of dosage compensation may be independent of the expression of XIST induced XC inactivation. Our study describes molecular landmarks of embryogenesis in pig that will provide a better strategy for derivation of porcine PSCs and improve research in regenerative medicine.

Aberrant epigenetic reprogramming is the main obstacle to the development of somatic cell nuclear transfer (SCNT) embryos and the generation of induced pluripotent stem (iPS) cells, which results in the low reprogramming efficiencies of SCNT and iPS. Histone H3 lysine 27 trimethylation (H3K27me3), as a repressive epigenetic mark, plays important roles in mammalian development and iPS induction. However, the reprogramming of H3K27me3 in pig remains elusive. In this study, we showed that H3K27me3 levels in porcine early cloned embryos were higher than that in IVF embryos. Then GSK126 and GSK-J4, two small molecule inhibitors of H3K27me3 methylase (EZH2) and demethylases (UTX/JMJD3), were used to regulate the H3K27me3 level. The results showed that H3K27me3 level was reduced in cloned embryos after treatment of PEF with 0.75 mM GSK126 for 48 h, incubation of one-cell reconstructed oocytes with 0.1 mM GSK126 and injection of antibody for EZH2 into oocyte. Meanwhile, the development of the cloned embryos was significantly improved after these treatments. On the contrary, GSK-J4 treatment increased the H3K27me3 level in cloned embryos and decreased the cloned embryonic development. Furthermore, iPS efficiency was both increased after reducing the H3K27me3 level in donor cells and in early reprogramming phase. In summary, our results suggest that H3K27me3 acts as an epigenetic barrier in SCNT and iPS reprogramming, and reduction of H3K27me3 level in donor cells and in early reprogramming phase can enhance both porcine SCNT and iPS efficiency.

The low success rate of somatic cell nuclear transfer (SCNT) in mammalian cloning is largely due to imprinting problems. However, little is known about the mechanisms of reprogramming imprinted genes during SCNT. Parental origin-specific DNA methylation regulates the monoallelic expression of imprinted genes. In natural fertilization, methylation imprints are established in the parental germline and maintained throughout embryonic development. However, it is unclear whether methylation imprints are protected from global changes of DNA methylation in cloned preimplantation embryos. Here, we demonstrate that cloned porcine preimplantation embryos exhibit demethylation at differentially methylated regions (DMRs) of imprinted genes; in particular, demethylation occurs during the first two cell cycles. By RNAi-mediated knockdown, we found that Dnmt1 is required for the maintenance of methylation imprints in porcine preimplantation embryos. However, no clear signals were detected in the nuclei of oocytes and preimplantation embryos by immunofluorescence. Thus, Dnmt1 is present at very low levels in the nuclei of porcine oocytes and preimplantation embryos and maintains methylation imprints. We further showed that methylation imprints were rescued in nonenucleated metaphase II (MII) oocytes. Our results indicate that loss of Dnmt1 in the maternal nucleus during SCNT significantly contributes to the unfaithful maintenance of methylation imprints in cloned embryos.

Derepressionof chromatin-mediated transcriptional repression of paternal and maternal genomes is considered the first major step that initiates zygotic gene expression after fertilization. The histone variant H3.3 is present in both male and female gametes and is thought to be important for remodeling the paternal and maternal genomes for activation during both fertilization and embryogenesis. However, the underlying mechanisms remain poorly understood. Using our H3.3B-HA-tagged mouse model, engineered to report H3.3 expression in live animals and to distinguish different source of H3.3 protein in embryos, we show here that sperm-derived H3.3 protein (sH3.3) is removed from the sperm genome shortly after fertilization and extruded from the zygotes via the second polar bodies (PB II) during embryogenesis. We also found that the maternal H3.3 (mH3.3) protein is incorporated into the paternal genome as early as 2h postfertilization and is detectable in the paternal genome until the morula stage. Knockdown of maternal H3.3 resulted in compromised embryonic development both of fertilized embryos and of androgenetic haploid embryos. Furthermore, we report that mH3.3 depletion in oocytes impairs both activation of the Oct4 pluripotency marker gene and global de novo transcription from the paternal genome important for early embryonic development. Our results suggest that H3.3-mediated paternal chromatin remodeling is essential for the development of preimplantation embryos and the activation of the paternal genome during embryogenesis. [1,2]. At the time of fertilization, both the sperm and oocyte genomes are transcriptionally repressed, whereas the maternal stored factors in the oocyte support and control the process of early embryogenesis. Maternal-to-zygotic transition (MZT) is an embryonic development stage under the exclusive control of the newly formed zygotic genome. This developmental process requires the zygotic genome activation (ZGA) to allow for the transition from specified germ cells to a totipotent embryo, in which both paternal and maternal genomes undergo dramatic epigenetic reprogramming regulated by maternal factors [3]. Overcoming chromatin-mediated transcriptional repression of paternal and maternal genomes is thought to be the first major step to initiate zygotic gene expression after fertilization [4][5][6][7][8][9].The mammalian sperm genome is packaged into highly condensed chromatin consisting primarily of protamine but 5-15% residual histones. Following fertilization, ZGA occurs first in the paternal genome (male pronucleus) at the 1-cell stage embryo, while activation of the maternal genome is usually delayed and occurs at the 2-cell stage in mice [10][11][12][13]. Soon after fertilization, protamine is removed from the sperm genome. The paternal genomes subsequently undergoes chromatin remodeling through a massive and highly regulated exchange of canonical and variant histones including H1FOO, H3.3, microH2A and H2A.Z. The incorporation of histone variants into paternal genome is ...